Nitrogen-containing heteroaryl compounds and methods of use thereof

ABSTRACT

The present invention relates to compounds suitable for use in mediating hypoxia inducible factor and for treating erythropoietin-associated conditions by increasing endogenous erythropoietin in vitro and in vivo.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation Patent Application of U.S. Utilitypatent application Ser. No. 12/015,275, filed Jan. 16, 2008, now U.S.Pat. No. 8,017,625, which applications is a Divisional PatentApplication of U.S. Utility patent application Ser. No. 10/861,082,filed Jun. 4, 2004, now U.S. Pat. No. 7,323,475, which claims thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No.60/476,811, filed Jun. 6, 2003; No. 60/476,420 filed, Jun. 6, 2003; No.60/476,633, filed Jun. 6, 2003; and No. 60/476,519, filed Jun. 6, 2003;all of which are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods and compounds capable ofmodulating the stability of the alpha subunit of hypoxia induciblefactor (HIF) and increasing endogenous erythropoietin, ex vivo and invivo.

2. State of the Art

An early response to tissue hypoxia is induction of hypoxia induciblefactor (HIF), a basic helix-loop-helix (bHLH) PAS (Per/Amt/Sim)transcriptional activator that mediates changes in gene expression inresponse to changes in cellular oxygen concentration. HIF is aheterodimer containing an oxygen-regulated alpha subunit (HIFα) and aconstitutively expressed beta subunit (HIM, also known as arylhydrocarbon receptor nuclear transporter (ARNT). In oxygenated(normoxic) cells, HIFα subunits are rapidly degraded by a mechanism thatinvolves ubiquitination by the von Hippel-Lindau tumor suppressor (pVHL)E3 ligase complex. Under hypoxic conditions, HIFα is not degraded, andan active HIFα/β complex accumulates in the nucleus and activates theexpression of several genes including glycolytic enzymes, glucosetransporter (GLUT)-1, erythropoietin (EPO), and vascular endothelialgrowth factor (VEGF). (Jiang, et al., (1996) J. Biol. Chem.,271:17771-17778; Iliopoulus, et al., (1996) Proc. Natl. Acad. Sci. USA,93:10595-10599; Maxwell, et al., (1999), Nature, 399:271-275; Sutter, etal., (2000) Proc. Natl. Acad. Sci. USA, 97:4748-4753; Cockman, et al.,(2000) J. Biol. Chem., 275:25733-25741; and Tanimoto, et al., (2000)EMBO. J. 19:4298-4309.)

Levels of HIFα protein are elevated in most cells in response to hypoxiaand HIFα is induced in vivo when animals are subjected to anemia orhypoxia. HIFα levels rise within a few hours after the onset of hypoxiaand return to baseline under continued hypoxic conditions. HIF has beenimplicated in numerous cellular and developmental processes includingcell proliferation, angiogenesis, and cell cycle arrest. HIFα has alsobeen associated with myocardial acute ischemia and early infarction,pulmonary hypertension, and inflammation. Although HIFα has beenassociated with tumor growth and metastasis, there is little indicationthat HIF is directly involved in tumorigenesis. Hypoxic preconditioning,in which a target organ is subjected to brief periods of hypoxia, hasbeen shown to protect both myocardium and brain against hypoxic-ischemicinjury. HIFα stabilization is closely associated with ischemia and isinduced by preconditioning. (Wang and Semenza, (1993) Proc. Natl. Acad.Sci. USA, 90:4304-4308; Stroka, et al., (2001) FASEB. J., 15:2445-2453;Semenza, et al., (1997) Kidney Int., 51:553-555; Carmeliet, et al.,(1998), Nature 394:485-490; Zhong, et al., (1999) Cancer Res.,59:5830-5835; Lee, et al., (2000) N. Engl. J. Med., 343:148-149; Sharp,et al., (2000) J. Cereb. Blood Flow Metab., 20:1011-1032; Semenza, etal., (2000) Adv. Exp. Med. Biol., 475:123-130; Thornton, et al., (2000)Biochem. J. 350:307-312; Deindl and Schaper, (1998) Mol. Cell. Biochem.,186:43-51; Bergeron, et al., (2000) Ann. Neurol. 48:285-296.)

Several investigators have studied the mechanism of interaction betweenHIFα and pVHL. An oxygen-dependent degradation domain (ODD) withinHIF-1α from residue 401 to 603 was originally identified as sufficientto confer oxygen-dependent instability to chimeric protein constructs. Adomain containing a portion of the ODD, from residue 526 to 652, wasfound to be required for pVHL-dependent degradation. Further, mutationof P₅₆₄YI to aspartic acids or mutation of K₅₃₂ to arginine within aregion conserved among HIFα homologs (residue 556 to 574 in HIF-1α)rendered the full-length HIFα protein stable under normoxic conditionsand resistant to pVHL-mediated degradation. (Huang, et al., (1998) Proc.Natl. Acad. Sci. USA, 95:7987-7992; and Tanimoto, et al., (2000) EMBO.J. 19:4298-4309.)

HIFα levels are increased by a number of factors that mimic hypoxia,including iron chelators such as desferrioxamine (DFO) and divalentmetal salts such as CoCl₂. HIFα levels are increased by angiotensin II,thrombin, and platelet-derived growth factor under normoxic conditionsusing a mechanism involving reactive oxygen species. Reports have alsosuggested HIFα is regulated by phosphorylation through pathwaysinvolving nitric oxide-activated phosphatidylinositol 3″-kinase (PI3K),hepatocyte growth factor, or mitogen-activated protein kinase.Glycogen-synthase kinase, which is a downstream target of PI3K, directlyphosphorylates the HIFα ODD domain. (Richard, et al., (2000) J. Biol.Chem., 275:26765-26771; Sandau, et al., (2000) Biochem. Biophys. Res.Commun. 278:263-267; Tacchini, et al., (2001) Carcinogenesis,22:1363-1371; and Sodhi, et al., (2001) Biochem. Biophys. Res. Commun.,287:292-300.)

Erythropoietin (EPO), a naturally occurring hormone that is produced inresponse to HIFα, stimulates the production of red blood cells(erythrocytes), which carry oxygen throughout the body. EPO is normallysecreted by the kidneys, and endogenous EPO is increased underconditions of reduced oxygen (hypoxia). All types of anemia arecharacterized by the blood's reduced capacity to carry oxygen, and thusare associated with similar signs and symptoms, including pallor of theskin and mucous membranes, weakness, dizziness, easy fatigability, anddrowsiness, leading to a decrease in quality of life. Subjects withsevere cases of anemia show difficulty in breathing and heartabnormalities. Anemia is typically associated with a condition in whichthe blood is deficient in red blood cells or in hemoglobin.

Common causes of anemia include deficiencies of iron, vitamin B₁₂, andfolic acid. Anemia can also develop in association with chronicdiseases, e.g., in inflammatory disorders, including disorders withconsequent inflammatory suppression of marrow, etc. Anemia may be causedby loss of blood, for example, due to accidents, surgery, orgastrointestinal bleeding caused by medications such as aspirin andibuprofen. Excessive blood loss can also be seen in women with heavymenstrual periods, and in people with stomach ulcers, duodenal ulcers,hemorrhoids, or cancer of the stomach or large intestine, etc.

Various conditions can cause the destruction of erythrocytes(hemolysis), thus leading to anemia. For example, allergic-typereactions to bacterial toxins and various chemical agents such assulfonamides and benzene can cause hemolysis. Hemolytic anemia is oftencaused by chemical poisoning, parasites, infection, or sickle-cellanemia. In addition, there are unusual situations in which the bodyproduces antibodies against its own erythrocytes, resulting inhemolysis. Any disease or injury to the bone marrow can cause anemia,since that tissue is the site of erythropoiesis, i.e. erythrocytesynthesis. Irradiation, disease, or various chemical agents can alsocause bone marrow destruction, producing aplastic anemia. Cancerpatients undergoing chemotherapy often have aplastic anemia. Anemia isalso associated with renal dysfunction, the severity of the anemiacorrelating highly with the extent of the dysfunction. Most patientswith renal failure undergoing dialysis suffer from chronic anemia.

In addition to being produced in the kidney, erythropoietin is producedby astrocytes and neurons in the central nervous system (CNS), and EPOand EPO receptors are expressed at capillaries of the brain-peripheryinterface. Furthermore, systemically administered EPO crosses theblood-brain barrier and reduces neuronal cell loss in response tocerebral and spinal chord ischemia, mechanical trauma, epilepsy,excitotoxins, and neuroinflammation. (Sakanaka, (1998) Proc. Natl. Acad.Sci. USA, 95:4635-4640; Celik, et al., (2002) Proc. Natl. Acad. Sci.USA, 99:2258-2263; Brines, et al., (2000) Proc. Natl. Acad. Sci. USA,97:10526-10531; Calapai, et al., (2000) Eur. J. Pharmacol., 401:349-356;and Siren, et al., (2001) Proc. Natl. Acad. Sci. USA, 98:4044-404.)

In the late 1980s, Amgen introduced a genetically engineered EPO for thetreatment of anemia in chronic renal failure patients. EPO is alsoadministered to cancer patients undergoing radiation and/orchemotherapy, decreasing the need for blood transfusions. EPO is used totreat anemia associated with HIV infection or azidothymidine (AZT)therapy. Although the market for EPO therapy is increasing, future salesare adversely affected by the high cost of the product. In addition,recombinant EPO therapy requires intravenous administration of EPO oneto three times per week for up to twelve weeks, a treatment regimen thatlimits self-administration and is inconvenient for the patient. Further,human serum EPO shows size heterogeneity due to extensive and variedglycosylation not reproduced in any recombinant human EPO.

Hypoxia, the condition that induces the production of HIFα, is a stateof reduced oxygen, which can occur when the lungs are compromised orblood flow is reduced. Ischemia, reduction in blood flow, can be causedby the obstruction of an artery or vein by a blood clot (thrombus) or byany foreign circulating matter (embolus), or by a vascular disorder suchas atherosclerosis. Reduction in blood flow can have a sudden onset andshort duration (acute ischemia), or can have a slow onset with longduration or frequent recurrence (chronic ischemia). Acute ischemia isoften associated with regional, irreversible tissue necrosis (aninfarct), whereas chronic ischemia is usually associated with transienthypoxic tissue injury. If the decrease in perfusion is prolonged orsevere, however, chronic ischemia can also be associated with aninfarct. Infarctions commonly occur in the spleen, kidney, lungs, brain,and heart, producing disorders such as intestinal infarction, pulmonaryinfarction, ischemic stroke, and myocardial infarction.

Pathologic changes in ischemic disorders depend on the duration andseverity of ischemia, and on the length of patient survival. Necrosiscan be seen within the infarct in the first 24 hours, and an acuteinflammatory response develops in the viable tissue adjacent to theinfarct with leukocytes migrating into the area of dead tissue. Oversucceeding days, there is a gradual breakdown and removal of cellswithin the infarct by phagocytosis, and replacement with a collagenousor glial scar.

Hypoperfusion or infarction in one organ often affects other organs. Forexample, ischemia of the lung, caused by, for example, a pulmonaryembolism, not only affects the lung, but also puts the heart and otherorgans, such as the brain, under hypoxic stress. Myocardial infarction,which often involves coronary artery blockage due to thrombosis,arterial wall vasospasms, or viral infection of the heart, can lead tocongestive heart failure and systemic hypotension. Secondarycomplications such as global ischemic encephalopathy can develop if thecardiac arrest is prolonged with continued hypoperfusion. Cerebralischemia, most commonly caused by vascular occlusion due toatherosclerosis, can range in severity from transient ischemic attacks(TIAs) to cerebral infarction or stroke. While the symptoms of TIAs aretemporary and reversible, TIAs tend to recur and are often followed by astroke.

Occlusive arterial disease includes coronary artery disease, which canlead to myocardial infarction, and peripheral arterial disease, whichcan affect the abdominal aorta, its major branches, and arteries of thelegs. Peripheral arterial disease includes Buerger's disease, Raynaud'sdisease, and acrocyanosis. Although peripheral arterial disease iscommonly caused by atherosclerosis, other major causes include, e.g.,diabetes, etc. Complications associated with peripheral arterial diseaseinclude severe leg cramps, angina, abnormal heart rhythms, heartfailure, heart attack, stroke, and kidney failure.

Ischemic and hypoxic disorders are a major cause of morbidity andmortality. Cardiovascular diseases cause at least 15 million deathsevery year and are responsible for 30% of deaths worldwide. Among thevarious cardiovascular diseases, ischemic heart disease andcerebrovascular diseases cause approximately 17% of deaths. Annually,1.3 million cases of nonfatal acute myocardial infarction are reported,making the prevalence approximately 600 per 100,000 people. Further, anestimated five million Americans suffer from venous thrombosis everyyear, and approximately 600,000 of these cases result in pulmonaryembolism. About one-third of the pulmonary embolisms end in death,making pulmonary embolism the third most common cause of death in theUnited States.

Currently, treatment of ischemic and hypoxic disorders is focused onrelief of symptoms and treatment of causative disorders. For example,treatments for myocardial infarction include nitroglycerin andanalgesics to control pain and relieve the workload of the heart. Othermedications, including digoxin, diuretics, aminone, β-blockers,lipid-lowering agents and angiotensin-converting enzyme inhibitors, areused to stabilize the condition, but none of these therapies directlyaddress the tissue damage produced by the ischemia and hypoxia.

Due to deficiencies in current treatments and in the production and useof recombinant EPO, there remains a need for compounds that areeffective in treating erythropoietin-associated conditions such asanemia, including anemia associated with diabetes, ulcers, kidneyfailure, cancer, infection, dialysis, surgery, and chemotherapy andconditions involving ischemia and hypoxia such as occlusive arterialdisease, angina pectoris, intestinal infarctions, pulmonary infarctions,cerebral ischemia, and myocardial infarction. There is also a need forcompounds that are effective in the prevention of tissue damage causedby ischemia that occurs due to, e.g., atherosclerosis, diabetes, andpulmonary disorders such as pulmonary embolism and the like. In summary,there is a need in the art for methods and compounds that modulate HIFand/or endogenous erythropoietin and can be used to treat and preventHIF-associated and EPO-associated disorders including conditionsinvolving anemia, ischemia and hypoxia.

SUMMARY OF THE INVENTION

This invention is directed to novel compounds and methods that canmodulate hypoxia inducible factor (HIF) and/or endogenous erythropoietin(EPO).

In one of its compound aspects, there is provided compounds representedby formula I:

wherein:

q is zero or one;

p is zero or one;

R^(a) is —COOH or —WR⁸; provided that when R^(a) is —COOH then p is zeroand when R^(a) is —WR⁸ then p is one;

W is selected from the group consisting of oxygen, —S(O)_(n)— and —NR⁹—where n is zero, one or two, R⁹ is selected from the group consisting ofhydrogen, alkyl, substituted alkyl, acyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic and R⁸ is selected from the group consisting of hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic, orwhen W is —NR⁹— then R⁸ and R⁹, together with the nitrogen atom to whichthey are bound, can be joined to form a heterocyclic or a substitutedheterocyclic group, provided that when W is —S(O)_(n)— and n is one ortwo, then R⁸ is not hydrogen;

R¹ is selected from the group consisting of hydrogen, alkyl, substitutedalkyl, alkoxy, substituted alkoxy, amino, substituted amino, aminoacyl,aryl, substituted aryl, halo, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic, and —XR⁶ where X is oxygen,—S(O)_(n)— or —NR⁷— where n is zero, one or two, R⁶ is selected from thegroup consisting of alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic, and R⁷ is hydrogen, alkyl or aryl or, when X is —NR⁷—,then R⁷ and R⁶, together with the nitrogen atom to which they are bound,can be joined to form a heterocyclic or substituted heterocyclic group;

R² and R³ are independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, halo, hydroxy, cyano, —S(O)_(n)—N(R⁶)—R⁶ where nis 0, 1, or 2, —NR⁶C(O)NR⁶R⁶, —XR⁶ where X is oxygen, —S(O)_(n)— or—NR⁷— where n is zero, one or two, each R⁶ is independently selectedfrom the group consisting of hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclicprovided that when X is —SO— or —SO₂—, then R⁶ is not hydrogen, and R⁷is selected from the group consisting of hydrogen, alkyl, aryl, or R²,R³ together with the carbon atom pendent thereto, form an arylsubstituted aryl, heteroaryl, or substituted heteroaryl;

R⁴ and R⁵ are independently selected from the group consisting ofhydrogen, halo, alkyl, substituted alkyl, alkoxy, substituted alkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl and —XR⁶where X is oxygen, —S(O)_(n)— or —NR⁷— where n is zero, one or two, R⁶is selected from the group consisting of alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic, and R⁷ is hydrogen, alkyl or aryl or, when Xis —NR⁷—, then R⁷ and R⁶, together with the nitrogen atom to which theyare bound, can be joined to form a heterocyclic or substitutedheterocyclic group;

R is selected from the group consisting of hydrogen, deuterium andmethyl;

R′ is selected from the group consisting of hydrogen, deuterium, alkyland substituted alkyl; alternatively, R and R′ and the carbon pendentthereto can be joined to form cycloalkyl, substituted cycloalkyl,heterocyclic or substituted heterocyclic group;

R″ is selected from the group consisting of hydrogen and alkyl or R″together with R′ and the nitrogen pendent thereto can be joined to forma heterocyclic or substituted heterocyclic group;

R′″ is selected from the group consisting of hydroxy, alkoxy,substituted alkoxy, acyloxy, cycloalkoxy, substituted cycloalkoxy,aryloxy, substituted aryloxy, heteroaryloxy, substituted heteroaryloxy,aryl, —S(O)_(n)—R¹⁰ wherein R¹⁰ is selected from the group consisting ofalkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl and substituted heteroaryl and n is zero,one or two;

and pharmaceutically acceptable salts, esters and prodrugs thereof;

with the proviso that when R, R′ and R″ are hydrogen and q is zero, andR^(a) is either —COOH (p is zero) or —WR⁸ (p is one) and W is oxygen andR⁸ is hydrogen then at least one of the following occurs:

1) R¹ is fluoro, bromo, iodo, alkyl, substituted alkyl, alkoxy,aminoacyl, substituted alkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, substituted heterocyclic, and —XR⁶where X is oxygen, —S(O)_(n)— or —NR⁷— where n is zero, one or two, R⁶is selected from the group consisting of alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic, and R⁷ is hydrogen, alkyl or aryl; or

2) R² is substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, fluoro, bromo, iodo, cyano, —XR⁶ where X isoxygen, —S(O)_(n)— or —NR⁷— where n is zero, one or two, R⁶ is selectedfrom the group consisting of alkyl, substituted alkyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic, and R⁷ is hydrogen, alkyl or aryl provided that:

a) when R² is substituted alkyl such a substituent does not includetrifluoromethyl;

b) —XR⁶ is not alkoxy; and

c) when —XR⁶ is substituted alkoxy such a substituent does not includebenzyl or benzyl substituted by a substituent selected from the groupconsisting of (C₁-C₅) alkyl and (C₁-C₅) alkoxy or does not include afluoroalkoxy substituent of the formula:—O—[CH₂]_(x)—C_(f)H_((2f+1−g))F_(g)

-   -   where x is zero or one; f is an integer of from 1 to 5; and g is        an integer of from 1 to (2f+1); or

3) R³ is substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, bromo, iodo, —XR⁶ where X is oxygen, —S(O)_(n)—or —NR⁷— where n is zero, one or two, R⁶ is selected from the groupconsisting of alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic, and R⁷ is hydrogen, alkyl or aryl provided that:

a) when R³ is substituted alkyl such a substituent does not includetrifluoromethyl;

b) —XR⁶ is not alkoxy; and

c) when —XR⁶ is substituted alkoxy such a substituent does not includebenzyl or benzyl substituted by a substituent selected from the groupconsisting of (C₁-C₅) alkyl and (C₁-C₅) alkoxy or does not include afluoroalkoxy substituent of the formula:—O—[CH₂]_(x)—C_(f)H_((2f+1−g))F_(g)

-   -   where x is zero or one; f is an integer of from 1 to 5; and g is        an integer of from 1 to (2f+1); or

4) R⁴ is iodo, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, —XR⁶ where X is oxygen, —S(O)_(n)— or —NR⁷—where n is zero, one or two, R⁶ is selected from the group consisting ofalkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic, andR⁷ is hydrogen, alkyl or aryl provided that:

a) when R⁴ is substituted alkyl such a substituent does not includetrifluoromethyl;

b) —XR⁶ is not alkoxy; and

c) when —XR⁶ is substituted alkoxy such a substituent does not include afluoroalkoxy substituent of the formula:—O—[CH₂]_(x)—C_(f)H_((2f+1−g))F_(g)

where x is zero or one; f is an integer of from 1 to 5; and g is aninteger of from 1 to (2f+1); or

5) R⁵ is iodo, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, —XR⁶ where X is oxygen, —S(O)_(n)— or —NR⁷—where n is zero, one or two, R⁶ is selected from the group consisting ofalkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic, andR⁷ is hydrogen, alkyl or aryl provided that:

a) when R⁵ is substituted alkyl such a substituent does not includetrifluoromethyl;

b) —XR⁶ is not alkoxy; and

c) when —XR⁶ is substituted alkoxy such a substituent does not include afluoroalkoxy substituent of the formula:—O—[CH₂]_(x)—C_(f)H_((2f+1−g))F_(g)

where x is zero or one; f is an integer of from 1 to 5; and g is aninteger of from 1 to (2f+1);

and with the further following proviso:

that when R¹, R³, R⁴, and R⁵ are hydrogen, then R² is not bromo.

In an alternative embodiment, the compounds of formula I are representedby formula IA:

wherein R¹, R², R³, R⁴, R⁵, R, R′, R″, R′″ and q are as defined above;and

pharmaceutically acceptable salts, esters, prodrugs thereof.

In an another alternative embodiment, the compounds of formula I arerepresented by the formula IB:

wherein R¹, R², R³, R⁴, R⁵, R″, R′″, WR⁸ and q are as defined above; and

pharmaceutically acceptable salts, esters, prodrugs thereof.

In an another alternative embodiment, the invention is directed tocompounds represented by the formula IC:

wherein R¹, R², R³, R⁴, R⁵, R, R′, R″, R′″, WR⁸ and q are as definedabove; and

pharmaceutically acceptable salts, esters, prodrugs thereof.

In yet another alternative embodiment, the invention is directed tocompounds represented by the formula ID:

wherein R¹, R², R³, R⁴, R⁵, R, R′, R″, R′″ and q are as defined above;and

pharmaceutically acceptable salts, esters, prodrugs thereof.

In other embodiments, the invention is directed to compounds representedby the formulae IIA, IIB, IIC, and IID, wherein said formulae aredefined below.

Preferred Embodiments

In compounds of formulae I, IA, IB, IC, and ID, preferably R¹ isselected from the group consisting of hydrogen, alkyl, substitutedalkyl, halo, alkoxy, aryloxy, substituted aryloxy, substituted aryl,alkylthio, aminoacyl, aryl, substituted amino, heteroaryl,heteroaryloxy, —S(O)_(n)-aryl, —S(O)_(n)-substituted aryl,—S(O)_(n)-heteroaryl, and —S(O)_(n)-substituted heteroaryl, where n iszero, one or two.

More preferably, R′ is selected from the group consisting of:

-   -   (3-methoxyphenyl)sulfanyl;    -   (4-chlorophenyl)sulfanyl;    -   (4-methylphenyl)sulfanyl;    -   2-fluorophenoxy;    -   2-methoxyphenoxy;    -   (2-methoxyphenyl)sulfanyl    -   3-fluorophenoxy;    -   3-methoxyphenoxy;    -   4-(methylcarbonylamino)phenoxy;    -   4-(methylsulfonamido)phenoxy;    -   4-fluorophenoxy;    -   4-methoxyphenoxy;    -   4-methoxyphenylsulfanyl;    -   4-methylphenyl;    -   bromo;    -   chloro;    -   dimethylaminomethyl;    -   ethoxy;    -   ethylsulfanyl;    -   hydrogen;    -   isopropyl;    -   methoxy;    -   methoxymethyl;    -   methyl;    -   N,N-dimethylaminocarbonyl;    -   naphth-2-yloxy;    -   naphthylsulfanyl;    -   phenoxy;    -   phenyl;    -   phenylamino;    -   phenylsulfinyl;    -   phenylsulfanyl;    -   pyridin-2-yloxy;    -   pyridin-2-yl; and    -   pyridin-2-ylsulfanyl.

In compounds of formulae I, IA, IB, IC and ID, R² is preferably selectedfrom the group consisting of substituted amino, aryloxy, substitutedaryloxy, alkoxy, substituted alkoxy, halo, hydrogen, alkyl, substitutedalkyl, aryl, —S(O)_(n)-aryl, —S(O)_(n)-substituted aryl,—S(O)_(n)-cycloalkyl, where n is zero, one or two, aminocarbonylamino,heteroaryloxy, and cycloalkyloxy.

More preferably, R² is selected from the group consisting of:

-   -   (4-methoxy)phenylsulfonylamino;    -   2,6-dimethylphenoxy;    -   3,4-difluorophenoxy;    -   3,5-difluorophenoxy;    -   3-chloro-4-fluorophenoxy;    -   3-methoxy-4-fluorophenoxy;    -   3-methoxy-5-fluorophenoxy;    -   4-(methylsulfonamido)phenoxy;    -   4-(phenylsulfonamido)phenoxy;    -   4-CF₃—O-phenoxy;    -   4-CF₃-phenoxy;    -   4-chlorophenoxy;    -   4-fluorophenoxy;    -   4-(4-fluorophenoxy)phenoxy;    -   4-methoxyphenoxy;    -   4-nitrophenoxy;    -   benzyloxy;    -   bromo;    -   butoxy;    -   CF₃;    -   chloro;    -   cyclohexyloxy;    -   cyclohexylsulfanyl;    -   cyclohexylsulfonyl;    -   fluoro;    -   hydrogen;    -   iodo;    -   isopropoxy;    -   methyl;    -   phenoxy;    -   phenyl;    -   phenylsulfanyl;    -   phenylsulfinyl;    -   phenylsulfonyl;    -   phenylurea;    -   pyridin-1-ylsulfanyl;    -   pyridin-3-yloxy; and    -   pyridin-4-ylsulfanyl.

In compounds of formulae I, IA, IB, IC, and ID, R³ is preferablyselected from the group consisting of: substituted aryloxy, substitutedalkoxy, alkoxy, substituted alkyl, alkyl, amino, cycloalkyloxy,hydrogen, halo, aryl, —S(O)_(n)-aryl, —S(O)_(n)-substituted aryl,—S(O)_(n)-heteroaryl, and —S(O)_(n)-substituted heteroaryl, where n iszero, one or two, aminocarbonylamino, and heteroaryloxy.

More preferably, R³ is selected from the group consisting of:

-   -   amino;    -   (4-methyl)phenylsulfonylaminophenoxy;    -   3,4-difluorophenoxy;    -   3,5-difluorophenoxy;    -   3-fluoro-5-methoxy-phenoxy;    -   3-chloro-4-fluorophenoxy    -   4-CF₃—O-phenoxy;    -   4-CF₃-phenoxy;    -   4-chlorophenoxy;    -   4-fluorophenoxy;    -   4-(4-fluorophenoxy)phenoxy;    -   4-methoxyphenoxy;    -   benzyloxy;    -   bromo;    -   butoxy;    -   CF₃;    -   chloro;    -   cyclohexyloxy;    -   hydrogen;    -   iodo;    -   isopropoxy;    -   phenoxy;    -   phenyl;    -   phenylsulfanyl;    -   phenylsulfonyl;    -   phenylsulfinyl;    -   phenylurea;    -   pyridin-1-ylsulfanyl;    -   pyridin-3-yloxy; and    -   pyridin-4-ylsulfanyl.

Alternatively, R² and R³, combined with the carbon atoms pendentthereto, are joined to form an aryl group. Preferably, the aryl group isphenyl.

In compounds of formulae I, IA, IB, IC, and ID, R⁴ is preferablyselected from the group consisting of: substituted arylthio, halo,hydrogen, substituted alkyl and aryl.

More preferably, R⁴ is selected from the group consisting of:

-   -   4-chlorophenyl sulfanyl;    -   chloro;    -   hydrogen;    -   methoxymethyl; and    -   phenyl.

In compounds of formulae I, IA, IB, IC, and ID, R⁵ is preferablyhydrogen or aryl. More preferably R⁵ is hydrogen or phenyl.

In compounds of formulae I, IA and IC, R is preferably selected from thegroup consisting of hydrogen, deuterium, aryl and alkyl. More preferablyR is selected from the group consisting of phenyl, hydrogen, deuteriumand methyl.

In compounds of formulae I, IA and IC, R′ is selected from the groupconsisting of preferably hydrogen, deuterium, alkyl, substituted alkyl,and substituted amino. More preferably, R′ is selected from the groupconsisting of:

-   -   4-aminobutyl;    -   4-hydroxybenzyl;    -   benzyl;    -   carboxylmethyl;    -   deuterium;    -   hydroxymethyl;    -   imidazol-4-ylmethyl;    -   isopropyl;    -   methyl; and    -   propyl.

Alternatively, R, R′ and the carbon atom pendent thereto join to form acycloalkyl and more preferably cyclopropyl.

In compounds of formulae I, IA, and IC, R″ is preferably hydrogen, alkylor substituted alkyl. More preferably, R″ is hydrogen, methyl orcarboxylmethyl (—CH₂C(O)OH). Alternatively, R′, R″ and the carbon atomand nitrogen atom respectively pendent thereto join to form aheterocyclic group and more preferably pyrrolidinyl.

In compounds of formulae I, IA, IB, IC, and ID, preferably R′″ isselected from the group consisting of hydrogen, hydroxy, alkoxy,substituted alkoxy, cycloalkoxy, substituted cycloalkoxy, thiol, acyloxyand aryl. Preferably, R′″ is selected from the group consisting of:

-   -   hydroxy;    -   benzyloxy;    -   ethoxy;    -   thiol;    -   methoxy;    -   methylcarbonyloxy; and    -   phenyl.

In compounds of formulae I, IB, and IC, WR⁸ is preferably selected fromthe group consisting of amino, substituted amino, aminoacyl, hydroxy,and alkoxy. More preferably, WR⁸ is selected from the group consistingof:

-   -   amino;    -   dimethylamino;    -   hydroxy;    -   methoxy; and    -   methylcarbonylamino.

Representative compounds for this application are presented in TablesA-D, wherein said table letter corresponds to formula letter (i.e.,representative compounds of formula IA are in Table A).

TABLE A

No. R¹ R² R³ R R′ R″  1 Cl H benzyloxy H methyl H  2 Cl H H Hhydroxymethyl H  3 Cl H H H hydroxymethyl H  4 Cl H isopropoxy Hhydroxymethyl H  5 Cl H isopropoxy H hydroxymethyl H  6 Cl isopropoxy HH hydroxymethyl H  7 Cl isopropoxy H H hydroxymethyl H  8 Cl H H methylmethyl H  9 Cl H isopropoxy methyl methyl H 10 Cl H H Himidazol-4-ylmethyl H 11 Cl H H H imidazol-4-ylmethyl H 12 Cl H H Hisopropyl H 13 Cl H H H isopropyl H 14 Cl H isopropoxy H isopropyl H 15Cl H isopropoxy H isopropyl H 16 Cl isopropoxy H H isopropyl H 17 Clisopropoxy H H isopropyl H 18 Cl H benzyloxy H isopropyl H 19 Cl H H Hbenzyl H 20 Cl H H H benzyl H 21 Cl H isopropoxy H benzyl H 22 Cl Hisopropoxy H benzyl H 23 Cl isopropoxy H H benzyl H 24 Cl isopropoxy H Hbenzyl H 25 Cl H H H 4-hydroxybenzyl H 26 Cl H H H 4-hydroxybenzyl H 27Cl H isopropoxy H 4-hydroxybenzyl H 28 Cl H isopropoxy H 4-hydroxybenzylH 29 Cl isopropoxy H H 4-hydroxybenzyl H 30 Cl isopropoxy H H4-hydroxybenzyl H 31 Cl H isopropoxy H propyl H 32 Cl H isopropoxy Hpropyl H 33 Cl H H H R′ and R″ and the — carbon and nitrogen atomrespectively pendent to which R″ is attached join to form a pyrrolidinyl34 Cl H H H R′ and R″ and the — carbon and nitrogen atom respectivelypendent to which R″ is attached join to form a pyrrolidinyl 35 Cl Hisopropoxy H R′ and R″ and the — carbon and nitrogen atom respectivelypendent to which R″ is attached join to form a pyrrolidinyl 36 Cl Hisopropoxy H R′ and R″ and the — carbon and nitrogen atom respectivelypendent to which R″ is attached join to form a pyrrolidinyl 37 Cl H H H4-aminobutyl H 38 Cl H H H 4-aminobutyl H 39 Cl H isopropoxy H4-aminobutyl H 40 Cl H isopropoxy H 4-aminobutyl H 41 Cl isopropoxy H H4-aminobutyl H 42 Cl isopropoxy H H 4-aminobutyl H 43 Cl H H Hcarboxylmethyl H 44 Cl H H H carboxylmethyl H 45 Cl H isopropoxy Hcarboxylmethyl H 46 Cl H isopropoxy H carboxylmethyl H 47 Cl isopropoxyH H carboxylmethyl H 48 Cl H H — R, R′ together with H the carbon towhich they are attached join to form cyclopropyl 49 Cl H isopropoxy — R,R′ together with H the carbon to which they are attached join to formcyclopropyl 50 Cl H H D D H 51 Cl H benzyloxy H methyl H 52 Cl benzyloxyH H methyl H 53 Cl benzyloxy H H methyl H 54 Cl H H H methyl H 55 Cl H HH methyl H 56 Cl H isopropoxy H methyl H 57 Cl H isopropoxy H methyl H58 Cl isopropoxy H H methyl H 59 Cl isopropoxy H H methyl H 60 H4-chlorophenoxy H H methyl H 61 H H 4-chlorophenoxy H methyl H 62 H3,4-difluorophenoxy H H methyl H 63 H phenylsulfanyl H H methyl H 64 Hphenylsulfanyl H H methyl H 65 H phenoxy H H methyl H 66 H4-methoxyphenoxy H H methyl H 67 H phenylsulfonyl H H methyl H 68methoxymethyl phenoxy H H methyl H 69 methoxymethyl phenoxy H H methyl H70 H phenoxy H H methyl H 71 4-chlorophenyl sulfanyl H H H methyl H 724-chlorophenyl sulfanyl H H H methyl H 73 H 3-methoxy-4-fluorophenoxy HH methyl H 74 H cyclohexyloxy H H methyl H 75 methyl 4-fluorophenoxy H Hmethyl H 76 H 4-fluorophenoxy H H methyl H 77 methyl phenoxy H H methylH 78 methyl phenylsulfanyl H H methyl H 79 H 4-trifluoromethyl-phenoxy HH methyl H

TABLE B

No. R² R³ WR⁸  1 H H methoxy  2 isopropoxy H amino  3 H isopropoxymethoxy  4 H H amino  5 H H hydroxy  6 H isopropoxy hydroxy  7 H Hdimethylamino  8 H H methylcarbonylamino  9 H isopropoxy amino 10 Hisopropoxy dimethylamino 11 isopropoxy H methoxy 12 isopropoxy Hdimethylamino 13 isopropoxy H hydroxy

TABLE C

No. R² R³ 1 isopropoxy H 2 H isopropoxy 3 H H

TABLE D

No. R¹ R² R³ R⁴ R⁵ R″ R′′′  1 Br 2,6-di(CH₃)phenyloxy H H H H OH  2 Brbutoxy H H H H OH  3 Br phenoxy H H H H OH  4 Cl Br H H H H OH  5 Br ClH H H H OH  6 Cl I H H H H OH  7 Cl H I H H H OH  8 Cl phenoxy H H H HOH  9 Cl phenylsulfanyl H H H H OH  10 Br —CF₃ H H H H OH  11 Br Hphenoxy H H H OH  12 Cl H H phenyl H H OH  13 Cl 2,6-di(CH₃)phenyloxy HH H H OH  14 Br H CF₃ H H H OH  15 Br Br H H H H OH  16 Brphenylsulfanyl H H H H OH  17 Cl H phenylsulfanyl H H H OH  18 4-methoxyphenyl- H H H H H OH sulfanyl  19 Br H H phenyl H H OH  20 Cl phenyl H HH H OH  21 Br H H H H H OH  22 Br methyl H H H H OH  23 Br H butoxy H HH OH  24 Br H Cl H H H OH  25 Cl H phenoxy H H H OH  26 Br H phenoxy H HH OH  27 H I H H H H OH  28 Br phenyl H H H H OH  29 Br H phenyl H H HOH  30 ethyl sulfanyl H H H H H OH  31 phenoxy H H H H H OH  32 H Hphenyl H H H OH  33 Br H H H phenyl H OH  34 Br F H H H H OH  35 H2,6-di(CH₃)phenyloxy H H H H OH  36 Cl H phenyl H H H OH  37 H phenoxy HH H H OH  38 H phenylsulfanyl H H H H OH  39 H phenyl H H H H OH  40 H Hphenoxy H H H OH  41 H H phenylsulfanyl H H H OH  42 H H H phenyl H H OH 43 Cl H H H phenyl H OH  44 H H H H phenyl H OH  45 Cl F H H H H OH  46H F H H H H OH  47 H H Br H H H OH  48 H R²/R³ = phenyl — H H H OH  49Br H benzyloxy H H methyl OH  50 Cl H H H H methyl OH  51 Cl Hisopropoxy H H methyl OH  52 Cl isopropoxy H H H methyl OH  53 Cl H H HH CH₂COOH OH  54 Cl H isopropoxy H H CH₂COOH OH  55 naphth-2-yloxy H H HH H OH  56 pyridin-3-yloxy H H H H H OH  57 4-methoxy H H H H H OHphenoxy  58 3-methoxy H H H H H OH phenoxy  59 3-fluorophenoxy H H H H HOH  60 4-fluorophenoxy H H H H H OH  61 2-fluorophenoxy H H H H H OH  622-methoxy H H H H H OH phenoxy  63 4-(methyl carbonyl H H H H H OHamino) phenoxy  64 4-(methyl H H H H H OH sulfonamido) phenoxy  65phenyl amino H H H H H OH  66 H H pyridin-3-yloxy H H H OH  67 Hpyridin-3-yloxy H H H H OH  68 Cl H H H H H methoxy  69 Cl H H H H Hethoxy  70 methoxy H H H H H OH  71 ethoxy H H H H H OH  72 phenyl H H HH H methyl-carbonyloxy  73 phenyl H H H H H OH  74 ethoxy H H H H Hphenyl  75 Cl H H H H H phenyl  76 H H H H H H phenyl  77 methyl H H H HH OH  78 methoxy methyl H H H H H OH  79 N,N-dimethyl H H H H H OH aminocarbonyl  80 methyl H phenoxy H H H OH  81 methyl phenoxy H H H H OH  82methyl phenoxy H H H H benzyloxy  83 methyl phenoxy H H H H ethoxy  84N,N-dimethyl phenoxy H H H H OH amino carbonyl  85 methoxy methylphenoxy H H H H OH  86 4-methyl phenyl H H H H H OH  87 methyl 4-fluorophenoxy H H H H OH  88 Cl 4-methoxy phenoxy H H H H OH  89 H 4-methoxyphenoxy H H H H OH  90 Cl H 4-methoxy-phenoxy H H H OH  91 H H4-methoxy-phenoxy H H H OH  92 Cl 4-CF₃-phenoxy H H H H OH  93 H4-CF₃-phenoxy H H H H OH  94 Cl H 4-CF₃-phenoxy H H H OH  95 H H4-CF₃-phenoxy H H H OH  96 Cl 4-fluorophenoxy H H H H OH  97 H4-fluorophenoxy H H H H OH  98 Cl H 4-fluoro-phenoxy H H H OH  99 H H4-fluoro-phenoxy H H H OH 100 H pyridin-4-yl sulfanyl H H H H OH 101 H Hpyridin-4-yl sulfanyl H H H OH 102 H phenylsulfinyl H H H H OH 103 Hphenylsulfonyl H H H H OH 104 H H phenyl sulfinyl H H H OH 105 H Hphenyl sulfonyl H H H OH 106 H H amino H H H OH 107 H (4-methoxy)phenylsulfonyl H H H H OH amino 108 H phenylurea H H H H OH 109 H Hphenylurea H H H OH 110 phenyl sulfanyl H H H H H OH 111 (4-chlorophenyl) H H H H H OH sulfanyl 112 (4-methyl phenyl H H H H H OH sulfanyl113 pyridin-2- H H H H H OH ylsulfanyl 114 (3-methoxy phenyl) H H H H HOH sulfanyl 115 2-methoxy phenyl H H H H H OH sulfanyl 116 naphthylsulfanyl H H H H H OH 117 phenyl sulfinyl H H H H H OH 118 phenylsulfonyl H H H H H OH 119 H pyridin-2-yl sulfanyl H H H H OH 120 H Hpyridin-2-yl sulfanyl H H H OH 121 Cl phenoxy phenoxy H H H OH 122 Hphenoxy phenoxy H H H OH 123 H H (4-methyl)phenyl SO₂—NH- H H H OHphenoxy 124 H 4-nitrophenoxy H H H H OH 125 H phenoxy H H H H thiol 126H CF₃ H H H H thiol 127 H 4-(phenylsulfonamido) H H H H OH phenoxy 128 H4-(methylsulfonamido) H H H H OH phenoxy 129 H 4-chlorophenoxy H H H HOH 130 H H 4-chloro-phenoxy H H H OH 131 H H 3-fluoro-5-methoxy-phenoxyH H H OH 132 H 3-methoxy-5-fluorophenoxy H H H H OH 133 H3,4-difluorophenoxy H H H H OH 134 H H 3,4-difluoro-phenoxy H H H OH 135H 4-CF₃—O-phenoxy H H H H OH 136 H H 4-CF₃—O-phenoxy H H H OH 137 H3,5-difluorophenoxy H H H H OH 138 H H 3,5-difluorophenoxy H H H OH 139H 4-(4-fluorophenoxy) phenoxy H H H H OH 140 H H4-(4-fluorophenoxy)phenoxy H H H OH 141 H 3-chloro-4-fluorophenoxy H H HH OH 142 H H 3-chloro-4-fluorophenoxy H H H OH 143 methyl4-chlorophenoxy H H H H OH 144 methyl H 4-chlorophenoxy H H H OH 145methyl 3,5-difluorophenoxy H H H H OH 146 methyl 4-methoxy phenoxy H H HH OH 147 methyl H 4-methoxyphenoxy H H H OH 148 H H cyclohexyloxy H H HOH 149 H cyclohexyloxy H H H H OH 150 methyl cyclohexyloxy H H H H OH151 H cyclohexyl sulfanyl H H H H OH 152 H cyclohexyl sulfanyl H H H HOH 153 isopropyl H H H H H OH 154 pyridin-2-yl H H H H H OH 155 ethylphenoxy H H H H OH 156 dimethyl amino phenylsulfanyl H H H H OH methyl157 methyl phenylsulfanyl H H H H OH 158 methyl 4-trifluoromethylphenoxy H H H H OH

Compounds included within the scope of this invention include, forexample, those set forth below:

-   {[4-Hydroxy-1-(naphthalen-2-yloxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-1-(pyridin-3-yloxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-1-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-1-(3-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[1-(3-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[1-(4-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[1-(2-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-1-(2-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[1-(4-Acetylamino-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-1-(4-methanesulfonylamino-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   [(4-Hydroxy-1-phenylamino-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   {[4-Hydroxy-6-(pyridin-3-yloxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-7-(pyridin-3-yloxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   [(1-Chloro-4-methoxy-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(1-Chloro-4-ethoxy-isoquinoline-3-carbonyl-amino]-acetic acid;-   [(4-Hydroxy-1-methoxy-isoquinoline-3-carbonyl-amino]-acetic acid;-   [(1-Ethoxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(4-Acetoxy-1-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(4-Hydroxy-1-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(1-Ethoxy-4-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(1-Chloro-4-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(4-Phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(4-Hydroxy-1-methyl-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(4-Hydroxy-1-methoxymethyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Dimethylcarbamoyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Hydroxy-1-methyl-6-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Benzyloxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Ethoxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Dimethylcarbamoyl-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Hydroxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Hydroxy-1-p-tolyl-isoquinoline-3-carbonyl)-amino]-acetic acid;-   {[7-(4-Fluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[1-Chloro-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]amino}-acetic    acid;-   {[4-Hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[1-Chloro-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[1-Chloro-4-hydroxy-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[1-Chloro-4-hydroxy-6-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-6-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[1-Chloro-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[7-(4-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[1-Chloro-6-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[6-(4-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-7-(pyridin-4-ylsulfanyl)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-6-(pyridin-4-ylsulfanyl)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   [(7-Benzenesulfinyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(7-Benzenesulfonyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(6-Benzenesulfinyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(6-Benzenesulfonyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(6-Amino-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid;-   {[4-Hydroxy-7-(4-methoxy-benzenesulfonylamino)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-7-(3-phenyl-ureido)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-6-(3-phenyl-ureido)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   [(4-Hydroxy-1-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   {[1-(4-Chloro-phenylsulfanyl)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   [(4-Hydroxy-1-p-tolylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   {[4-Hydroxy-1-(pyridin-2-ylsulfanyl)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-1-(3-methoxy-phenylsulfanyl)-isoquinoline-3-carbonyl]amino}-acetic    acid;-   {[4-Hydroxy-1-(2-methoxy-phenylsulfanyl)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-1-(naphthalen-2-ylsulfanyl)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   [(1-Benzenesulfinyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Benzenesulfonyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   {[4-Hydroxy-7-(pyridin-2-ylsulfanyl)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-6-(pyridin-2-ylsulfanyl)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   [(1-Chloro-4-hydroxy-6,7-diphenoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Hydroxy-6,7-diphenoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   ({4-Hydroxy-7-[4-(toluene-4-sulfonylamino)-phenoxy]-isoquinoline-3-carbonyl}-amino)-acetic    acid;-   {[4-Hydroxy-7-(4-nitro-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   [(4-Mercapto-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(4-Mercapto-7-trifluoromethyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   {[7-(4-Benzenesulfonylamino-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-7-(4-methanesulfonylamino-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[7-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]amino}-acetic    acid;-   {[6-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[6-(3-Fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[7-(3-Fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[7-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[6-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-7-(4-trifluoromethoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-6-(4-trifluoromethoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   2-(S)-{[7-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionic    acid;-   2-(S)-{[6-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionic    acid;-   2-{[7-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionic    acid;-   2-(S)-[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   2-(R)-[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   2-(R)-[(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   2-(S)-{[4-Hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-propionic    acid;-   2-(S)-[(7-Benzenesulfonyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   (R)-2-[(4-Hydroxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   (S)-2-[(4-Hydroxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   (S)-2-[(4-Mercapto-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   (S)-2-{[1-(4-Chloro-phenylsulfanyl)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionic    acid;-   (R)-2-{[1-(4-Chloro-phenylsulfanyl)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionic    acid;-   [(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Chloro-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Chloro-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(4-Hydroxy-6-phenoxy-isoquinoline-3-carbonyl)-amino]acetic acid;-   [(1-Chloro-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Chloro-4-hydroxy-6-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-4-hydroxy-6-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   {[7-(2,6-Dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[1-Chloro-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[1-Bromo-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   [(1-Bromo-7-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-6-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-4-hydroxy-7-trifluoromethyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-4-hydroxy-6-trifluoromethyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Hydroxy-1-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(1,7-dibromo-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(7-Bromo-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(6-Bromo-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(1-Bromo-7-fluoro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(7-Fluoro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(1-Chloro-7-fluoro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Chloro-4-hydroxy-benzo[g]isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(4-Hydroxy-6-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(4-Hydroxy-7-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(1-Chloro-4-hydroxy-6-phenyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Chloro-4-hydroxy-7-phenyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-4-hydroxy-6-phenyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-4-hydroxy-7-phenyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(4-Hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(1-Chloro-4-hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Chloro-4-hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-4-hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-4-hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Ethylsulfanyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;    -   {[4-Hydroxy-1-(4-methoxy-phenylsulfanyl)-isoquinoline-3-carbonyl]-amino}-acetic        acid;-   [(1-Chloro-4-hydroxy-7-iodo-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Chloro-4-hydroxy-6-iodo-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Hydroxy-7-iodo-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(1-Bromo-4-hydroxy-7-methyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-7-butoxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Bromo-6-butoxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-methyl-amino]-acetic    acid;-   [(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-methyl-amino]-acetic    acid;-   [(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-methyl-amino]-acetic    acid;-   [(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-methyl-amino]-acetic    acid;-   [Carboxymethyl-(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [Carboxymethyl-(1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid    (2-amino-ethyl)-amide (trifluoro-acetic acid salt);-   1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid    (2-methoxy-ethyl)-amide;-   1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid    (2-hydroxy-ethyl)-amide;-   1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid    (2-dimethylamino-ethyl)-amide;-   1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid    (2-acetylamino-ethyl)-amide;-   1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylic acid    (2-hydroxy-ethyl)-amide;-   1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylic acid    (2-methoxy-ethyl)-amide;-   1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylic acid    (2-amino-ethyl)-amide (trifluoro-acetic acid salt);-   1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylic acid    (2-dimethylamino-ethyl)-amide;-   1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylic acid    (2-amino-ethyl)-amide (trifluoro-acetic acid salt);-   1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylic acid    (2-methoxy-ethyl)-amide;-   1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylic acid    (2-dimethylamino-ethyl)-amide;-   1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylic acid    (2-hydroxy-ethyl)-amide;-   (S)-2-[(6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   (R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-hydroxy-propionic    acid;-   (S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-hydroxy-propionic    acid;-   (R)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-hydroxy-propionic    acid;-   (S)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-hydroxy-propionic    acid;-   (R)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-hydroxy-propionic    acid;-   (S)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-hydroxy-propionic    acid;-   2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-2-methyl-propionic    acid;-   2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-2-methyl-propionic    acid;-   (R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-(1H-imidazol-4-yl)-propionic    acid (trifluoro-acetic acid salt);-   (S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-(1H-imidazol-4-yl)-propionic    acid (trifluoro-acetic acid salt);-   (R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyric    acid;-   (S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyric    acid;-   (R)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyric    acid;-   (S)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyric    acid;-   (R)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyric    acid;-   (S)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyric    acid;-   (S)-2-[(6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyric    acid;-   (R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-phenyl-propionic    acid;-   (S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-phenyl-propionic    acid;-   (R)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-phenyl-propionic    acid;-   (S)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-phenyl-propionic    acid;-   (R)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-phenyl-propionic    acid;-   (S)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-phenyl-propionic    acid;-   (R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-(4-hydroxy-phenyl)-propionic    acid;-   (S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-(4-hydroxy-phenyl)-propionic    acid;-   (R)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-(4-hydroxy-phenyl)-propionic    acid;-   (S)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-(4-hydroxy-phenyl)-propionic    acid;-   (R)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-(4-hydroxy-phenyl)-propionic    acid;-   (S)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-(4-hydroxy-phenyl)-propionic    acid;-   (R)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-pentanoic    acid;-   (S)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-pentanoic    acid;-   (R)-1-(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-pyrrolidine-2-carboxylic    acid;-   (S)-1-(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-pyrrolidine-2-carboxylic    acid;-   (R)-1-(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-pyrrolidine-2-carboxylic    acid;-   (S)-1-(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-pyrrolidine-2-carboxylic    acid;-   (R)-6-Amino-2-[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-hexanoic    acid (trifluoro-acetic acid salt);-   (S)-6-Amino-2-[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-hexanoic    acid (trifluoro-acetic acid salt);-   (R)-6-Amino-2-[(1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-hexanoic    acid; trifluoroacetic acid salt;-   (S)-6-Amino-2-[(1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-hexanoic    acid (trifluoro-acetic acid salt);-   (R)-6-Amino-2-[(1-chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-hexanoic    acid; trifluoroacetic acid salt;-   (S)-6-Amino-2-[(1-chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-hexanoic    acid (trifluoro-acetic acid salt);-   (R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-succinic    acid;-   (S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-succinic    acid;-   (R)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-succinic    acid;-   (S)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-succinic    acid;-   (R)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-succinic    acid;-   1-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-cyclopropanecarboxylic    acid;-   1-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-cyclopropanecarboxylic    acid;-   Dideutero-[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   (R)-2-[(6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   (S)-2-[(7-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   (R)-2-[(7-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   (S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   (R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   (S)-2-[(6-Isopropoxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   (R)-2-[6-Isopropoxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   (S)-2-[(7-Isopropoxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   (R)-2-[(7-Isopropoxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]propionic    acid;-   1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylic acid    (2-hydroxy-1-hydroxymethyl-ethyl)-amide;-   1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylic acid    (2-hydroxy-1-hydroxymethyl-ethyl)-amide;-   1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid    (2-hydroxy-1-hydroxymethyl-ethyl)-amide;-   {[7-(3,5-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[6-(3,5-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   ({7-[4-(4-Fluoro-phenoxy)-phenoxy]-4-hydroxy-isoquinoline-3-carbonyl}-amino)-acetic    acid;-   ({6-[4-(4-Fluoro-phenoxy)-phenoxy]-4-hydroxy-isoquinoline-3-carbonyl}-amino)-acetic    acid;-   {[7-(3-Chloro-4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[6-(3-Chloro-4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   (S)-2-{[7-(3-Fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionic    acid;-   2-(S)-[(7-Cyclohexyloxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   2-(S)-{[7-(4-Fluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-propionic    acid;-   2-(S)-{[7-(4-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionic    acid;-   2-(S)-[(4-Hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   2-(S)-[(4-Hydroxy-1-methyl-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-propionic    acid;-   2-(S)-{[4-Hydroxy-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-propionic    acid;-   {[7-(4-Chloro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[6-(4-Chloro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[7-(3,5-Difluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-7-(4-methoxy-phenoxy)-1-methyl-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   {[4-Hydroxy-6-(4-methoxy-phenoxy)-1-methyl-isoquinoline-3-carbonyl]-amino}-acetic    acid;-   [(6-Cyclohexyloxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(7-Cyclohexyloxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(7-Cyclohexyloxy-4-hydroxy-1-methyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(7-Cyclohexylsulfanyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(7-Cyclohexanesulfonyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Hydroxy-1-isobutyl-isoquinoline-3-carbonyl)-amino]-acetic acid;-   [(4-Hydroxy-1-pyridin-2-yl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Ethyl-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(1-Dimethylaminomethyl-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   [(4-Hydroxy-1-methyl-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic    acid;-   {[4-Hydroxy-1-methyl-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-acetic    acid; and

pharmaceutically acceptable salts, esters and prodrugs thereof.

In still another embodiment of the invention, a pharmaceuticalcomposition is provided comprising a pharmaceutically acceptableexcipient or carrier and a therapeutically effective amount of acompound of formula I or a mixture of such compounds.

Also provided are methods for treating, preventing or pretreating acondition mediated at least in part by HIF and/or EPO is provided. Themethod comprises administering to a mammalian patient a therapeuticallyeffective amount of a compound having the structure of formula I abovewith the proviso that the compound is not selected from the groupconsisting of:

-   N-((1-chloro-4-hydroxy-7-(2-propyloxy)    isoquinolin-3-yl)-carbonyl)-glycine,-   N-((1-chloro-4-hydroxy-6-(2-propyloxy)    isoquinolin-3-yl)-carbonyl)-glycine,-   N-((1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino) acetic acid,-   N-((1-chloro-4-hydroxy-7-methoxyisoquinolin-3-yl)-carbonyl)-glycine,-   N-((1-chloro-4-hydroxy-6-methoxyisoquinolin-3-yl)-carbonyl)-glycine,-   N-((7-butyloxy-1-chloro-4-hydroxyisoquinolin-3-yl)-carbonyl)-glycine,-   N-((6-benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino)-acetic    acid,-   N-((7-benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino)-acetic    acid,-   N-((8-chloro-4-hydroxyisoquinolin-3-yl)-carbonyl)-glycine,-   N-((7-butoxy-4-hydroxy-isoquinoline-3-carbonyl)-amino) acetic acid,    and-   ((7-benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)amino)acetic    acid methyl ester.

A further embodiment of this invention provides a method of inhibitingthe activity hydroxylase enzyme which modifies the alpha subunit ofhypoxia inducible factor.

This invention also contemplates a composition comprising the compoundof formula 1 or a mixture of compounds of formula 1 in combination withat least one additional therapeutic agent. Preferably, the additionaltherapeutic agent is erythropoietin.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that the invention is not limited to the particularmethodologies, protocols, cell lines, assays, and reagents described, asthese may vary. It is also to be understood that the terminology usedherein is intended to describe particular embodiments of the presentinvention, and is in no way intended to limit the scope of the presentinvention as set forth in the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural references unless thecontext clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications cited hereinare incorporated herein by reference in their entirety for the purposeof describing and disclosing the methodologies, reagents, and toolsreported in the publications that might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, cell biology, genetics, immunology and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.(See, e.g., Gennaro, A. R., ed. (1990) Remington's PharmaceuticalSciences, 18^(th) ed., Mack Publishing Co.; Colowick, S. et al., eds.,Methods In Enzymology, Academic Press, Inc.; Handbook of ExperimentalImmunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986,Blackwell Scientific Publications); Maniatis, T. et al., eds. (1989)Molecular Cloning: A Laboratory Manual, 2^(nd) edition, Vols. I-III,Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999)Short Protocols in Molecular Biology, 4^(th) edition, John Wiley & Sons;Ream et al., eds. (1998) Molecular Biology Techniques: An IntensiveLaboratory Course, Academic Press); PCR (Introduction to BiotechniquesSeries), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag)).

The term “anemia” as used herein refers to any abnormality in hemoglobinor erythrocytes that leads to reduced oxygen levels in the blood. Anemiacan be associated with abnormal production, processing, or performanceof erythrocytes and/or hemoglobin. The term anemia refers to anyreduction in the number of red blood cells and/or level of hemoglobin inblood relative to normal blood levels.

Anemia can arise due to conditions such as acute or chronic kidneydisease, infections, inflammation, cancer, irradiation, toxins,diabetes, and surgery. Infections may be due to, e.g., virus, bacteria,and/or parasites, etc. Inflammation may be due to infection, autoimmunedisorders, such as rheumatoid arthritis, etc. Anemia can also beassociated with blood loss due to, e.g., stomach ulcer, duodenal ulcer,hemorrhoids, cancer of the stomach or large intestine, trauma, injury,surgical procedures, etc. Anemia is further associated with radiationtherapy, chemotherapy, and kidney dialysis. Anemia is also associatedwith HIV-infected patients undergoing treatment with azidothymidine(zidovudine) or other reverse transcriptase inhibitors, and can developin cancer patients undergoing chemotherapy, e.g., with cyclic cisplatin-or non-cisplatin-containing chemotherapeutics. Aplastic anemia andmyelodysplastic syndromes are diseases associated with bone marrowfailure that result in decreased production of erythrocytes. Further,anemia can result from defective or abnormal hemoglobin or erythrocytes,such as in disorders including microcytic anemia, hypochromic anemia,etc. Anemia can result from disorders in iron transport, processing, andutilization, see, e.g., sideroblastic anemia, etc.

The terms “disorders,” “diseases,” and “conditions” are used inclusivelyand refer to any condition deviating from normal.

The terms “anemic conditions” and “anemic disorders” refer to anycondition, disease, or disorder associated with anemia. Such disordersinclude, but are not limited to, those disorders listed above. Anemicdisorders further include, but are not limited to, aplastic anemia,autoimmune hemolytic anemia, bone marrow transplantation, Churg-Strausssyndrome, Diamond Blackfan anemia, Fanconi's anemia, Felty syndrome,graft versus host disease, hematopoietic stem cell transplantation,hemolytic uremic syndrome, myelodysplastic syndrome, nocturnalparoxysmal hemoglobinuria, osteomyelofibrosis, pancytopenia, purered-cell aplasia, purpura Schoenlein-Henoch, sideroblastic anemia,refractory anemia with excess of blasts, rheumatoid arthritis, Shwachmansyndrome, sickle cell disease, thalassemia major, thalassemia minor,thrombocytopenic purpura, etc.

The term “erythropoietin-associated conditions” is used inclusively andrefers to any condition associated with below normal, abnormal, orinappropriate modulation of erythropoietin. Erythropoietin-associatedconditions include any condition wherein an increase in EPO level wouldprovide therapeutic benefit. Levels of erythropoietin associated withsuch conditions can be determined by any measure accepted and utilizedby those of skill in the art. Erythropoietin-associated conditionsinclude anemic conditions such as those described above.

Erythropoietin-associated conditions further include neurologicaldisorders and/or injuries, including cases of stroke, trauma, epilepsy,neurodegenerative disease and the like, wherein erythropoietin mayprovide a neuroprotective effect. Neurodegenerative diseasescontemplated by the invention include Alzheimer's disease, Parkinson'sdisease, Huntington's disease, and the like.

The term “erythropoietin” refers to any recombinant or naturallyoccurring erythropoietin including, e.g., human erythropoietin (GenBankAccession No. AAA52400; Lin et al. (1985) Proc Nat'l Acad. Sci. USA82:7580-7584), EPOETIN human recombinant erythropoietin (Amgen, Inc.,Thousand Oaks Calif.), ARANESP human recombinant erythropoietin (Amgen),PROCRIT human recombinant erythropoietin (Ortho Biotech Products, L.P.,Raritan N.J.), etc.

The term “HIFα” refers to the alpha subunit of hypoxia inducible factorprotein. HIFα may be any human or other mammalian protein, or fragmentthereof, including human HIF-1α (Genbank Accession No. Q16665), HIF-2α(Genbank Accession No. AAB41495), and HIF-3α (Genbank Accession No.AAD22668); murine HIF-1α (Genbank Accession No. Q61221), HIF-2α (GenbankAccession No. BAA20130 and AAB41496), and HIF-3α (Genbank Accession No.AAC72734); rat HIF-1α (Genbank Accession No. CAA70701), HIF-2α (GenbankAccession No. CAB96612), and HIF-3α (Genbank Accession No. CAB96611);and bovine HIF-1α (Genbank Accession No. BAA78675). HIFα may also be anynon-mammalian protein or fragment thereof, including Xenopus laevisHIF-1α (Genbank Accession No. CAB96628), Drosophila melanogaster HIF-1α(Genbank Accession No. JC4851), and chicken HIF-1α (Genbank AccessionNo. BAA34234). HIFα gene sequences may also be obtained by routinecloning techniques, for example by using all or part of a HIFα genesequence described above as a probe to recover and determine thesequence of a HIFα gene in another species.

A fragment of HIFα includes any fragment retaining at least onefunctional or structural characteristic of HIFα. Fragments of HIFαinclude, e.g., the regions defined by human HIF-1α from amino acids 401to 603 (Huang et al., supra), amino acid 531 to 575 (Jiang et al. (1997)J. Biol. Chem. 272:19253-19260), amino acid 556 to 575 (Tanimoto et al.,supra), amino acid 557 to 571 (Srinivas et al. (1999) Biochem BiophysRes. Commun 260:557-561), and amino acid 556 to 575 (Ivan and Kaelin(2001) Science 292:464-468). Further, HIFα fragments include anyfragment containing at least one occurrence of the motif LXXLAP, (SEQ IDNO:1) e.g., as occurs in the human HIF-1α native sequence at L₃₉₇TLLAP(SEQ ID NO:2) and L₅₅₉EMLAP (SEQ ID NO:3).

The terms “amino acid sequence” or “polypeptide” as used herein, e.g.,to refer to HIFα and fragments thereof, contemplate an oligopeptide,peptide, or protein sequence, or to a fragment of any of these, and tonaturally occurring or synthetic molecules. “Fragments” can refer to anyportion of a sequence that retains at least one structural or functionalcharacteristic of the protein. Immunogenic fragments or antigenicfragments are fragments of polypeptides, preferably, fragments of aboutfive to fifteen amino acids in length, that retain at least onebiological or immunological activity. Where “amino acid sequence” isused to refer to the polypeptide sequence of a naturally occurringprotein molecule, “amino acid sequence” and like terms are not meant tolimit the amino acid sequence to the complete native sequence associatedwith the recited protein molecule.

The term “related proteins” as used herein, for example, to refer toproteins related to HIFα prolyl hydroxylase, encompasses other2-oxoglutarate dioxygenase enzymes, especially those family members thatsimilarly require Fe²⁺, 2-oxoglutarate, and oxygen to maintainhydroxylase activity. Such enzymes include, but are not limited to,e.g., procollagen lysyl hydroxylase, procollagen prolyl 4-hydroxylase,and Factor Inhibiting HIF (FIH), an asparaginyl hydroxylase responsiblefor regulating transactivation of HIFα. (GenBank Accession No. AAL27308;Mahon et al. (2001) Genes Dev 15:2675-2686; Lando et al. (2002) Science295:858-861; and Lando et al. (2002) Genes Dev 16:1466-1471. See alsoElkins et al. (2002) J Biol Chem C200644200, etc.)

The terms “HIF prolyl hydroxylase” and “HIF PH” refer to any enzymecapable of hydroxylating a proline residue in the HIF protein.Preferably, the proline residue hydroxylated by HIF PH includes theproline found within the motif LXXLAP, (SEQ ID NO:1) e.g., as occurs inthe human HIF-1a native sequence at L₃₉₇TLLAP (SEQ ID NO:2) andL₅₅₉EMLAP (SEQ ID NO:3). HIF PH includes members of the Egl-Nine (EGLN)gene family described by Taylor (2001, Gene 275:125-132), andcharacterized by Aravind and Koonin (2001, Genome Biol 2: RESEARCH0007), Epstein et al. (2001, Cell 107:43-54), and Bruick and McKnight(2001, Science 294:1337-1340). Examples of HIF PH enzymes include humanSM-20 (EGLN1)(GenBank Accession No. AAG33965; Dupuy et al. (2000)Genomics 69:348-54), EGLN2 isoform 1 (GenBank Accession No. CAC42510;Taylor, supra), EGLN2 isoform 2 (GenBank Accession No. NP_(—)060025),and EGLN3 (GenBank Accession No. CAC42511; Taylor, supra); mouse EGLN1(GenBank Accession No. CAC42515), EGLN2 (GenBank Accession No.CAC42511), and EGLN3 (SM-20)(GenBank Accession No. CAC42517); and ratSM-20 (GenBank Accession No. AAA19321). Additionally, HIF PH may includeCaenorhabditis elegans EGL-9 (GenBank Accession No. AAD56365) andDrosophila melanogaster CG1114 gene product (GenBank Accession No.AAF52050). HIF PH also includes any fragment of the foregoingfull-length proteins that retain at least one structural or functionalcharacteristic.

The term “agonist” refers to a molecule that increases or prolongs theduration of the effect of a particular molecule. Agonists may includeproteins, nucleic acids, carbohydrates, or any other molecules thatincrease the effect(s) of the target molecule.

The term “antagonist” refers to a molecule that decreases the extent orduration of the effect of the biological or immunological activity of aparticular molecule. Antagonists may include proteins, nucleic acids,carbohydrates, antibodies, or any other molecules that decrease theeffect(s) of the target molecule.

The term “microarray” refers to any arrangement of nucleic acids, aminoacids, antibodies, etc., on a substrate. The substrate can be anysuitable support, e.g., beads, glass, paper, nitrocellulose, nylon, orany appropriate membrane, etc. A substrate can be any rigid orsemi-rigid support including, but not limited to, membranes, filters,wafers, chips, slides, fibers, beads, including magnetic or nonmagneticbeads, gels, tubing, plates, polymers, microparticles, capillaries, etc.The substrate can provide a surface for coating and/or can have avariety of surface forms, such as wells, pins, trenches, channels, andpores, to which the nucleic acids, amino acids, etc., may be bound.

The term “excipient” as used herein means an inert or inactive substanceused in the production of pharmaceutical products or other tablets,including without limitation any substance used as a binder,disintegrant, coating, compression/encapsulation aid, cream or lotion,lubricant, parenteral, sweetener or flavoring, suspending/gelling agent,or wet granulation agent. Binders include, e.g., carbopol, povidone,xanthan gum, etc.; coatings include, e.g., cellulose acetate phthalate,ethylcellulose, gellan gum, maltodextrin, etc.;compression/encapsulation aids include, e.g., calcium carbonate,dextrose, fructose dc, honey dc, lactose (anhydrate or monohydrate;optionally in combination with aspartame, cellulose, or microcrystallinecellulose), starch dc, sucrose, etc.; disintegrants include, e.g.,croscarmellose sodium, gellan gum, sodium starch glycolate, etc.; creamsand lotions include, e.g., maltodextrin, carrageenans, etc.; lubricantsinclude, e.g., magnesium stearate, stearic acid, sodium stearylfumarate, etc.; materials for chewable tablets include, e.g., dextrose,fructose dc, lactose (monohydrate, optionally in combination withaspartame or cellulose), etc.; parenterals include, e.g., mannitol,povidone, etc.; plasticizers include, e.g., dibutyl sebacate,polyvinylacetate phthalate, etc.; suspending/gelling agents include,e.g., carrageenan, sodium starch glycolate, xanthan gum, etc.;sweeteners include, e.g., aspartame, dextrose, fructose dc, sorbitol,sucrose dc, etc.; and wet granulation agents include, e.g., calciumcarbonate, maltodextrin, microcrystalline cellulose, etc.

The term “loading dose” as used herein refers to a single or multipledose administered initially to rapidly achieve the desiredpharmacological level. For example, a loading dose in reference to themethods of the invention refers to an initial dosing regimen thatrapidly increases, e.g., the plasma concentration of a compound of theinvention to a pharmaceutically active level.

The term “induction dose” as used herein refers to a repeated dosestrength administered initially to rapidly achieve the desiredphysiological response. For example, an induction dose in reference tothe methods of the invention refers to an initial dosing regimen thatrapidly increases the hematocrit or hemoglobin level to within a targetrange, which may be at or below normal hematocrit/hemoglobin levels.

The term “maintenance dose” as used herein refers to the dose leveladministered after a loading or induction dose in order to maintain adesired physiological response. For example, a maintenance dose inreference to the methods of the invention refers to a dosing regimenthat maintains hematocrit and/or hemoglobin within a desired targetrange, which may be at or below normal hematocrit/hemoglobin levels.

The term “sample” is used herein in its broadest sense. Samples may bederived from any source, for example, from bodily fluids, secretions,tissues, cells, or cells in culture including, but not limited to,saliva, blood, urine, serum, plasma, vitreous, synovial fluid, cerebralspinal fluid, amniotic fluid, and organ tissue (e.g., biopsied tissue);from chromosomes, organelles, or other membranes isolated from a cell;from genomic DNA, cDNA, RNA, mRNA, etc.; and from cleared cells ortissues, or blots or imprints from such cells or tissues. Samples may bederived from any source, such as, for example, a human subject, or anon-human mammalian subject, etc. Also contemplated are samples derivedfrom any animal model of disease. A sample can be in solution or can be,for example, fixed or bound to a substrate. A sample can refer to anymaterial suitable for testing for the presence of erythropoietin or HIFαor to fragments thereof, or suitable for screening for molecules thatincrease endogenous levels of erythropoietin or HIFα or to fragmentsthereof. Methods for obtaining such samples are within the level ofskill in the art.

The term “subject” is used herein in its broadest sense. Subjects mayinclude isolated cells, either prokaryotic or eukaryotic, or tissuesgrown in culture. In certain embodiments, a subject is an animal,particularly an animal selected from a mammalian species including rat,rabbit, bovine, ovine, porcine, canine, feline, murine, equine, andprimate, particularly human.

As used herein, “alkyl” refers to monovalent alkyl groups having from 1to 10 carbon atoms, preferably from 1 to 5 carbon atoms and morepreferably 1 to 3 carbon atoms. This term is exemplified by groups suchas methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, n-pentyl andthe like.

“Substituted alkyl” refers to an alkyl group, of from 1 to 10 carbonatoms, preferably, 1 to 5 carbon atoms, having from 1 to 5 substituents,preferably 1 to 3 substituents, independently selected from the groupconsisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aryl, substituted aryl,aryloxy, substituted aryloxy, aryloxyaryl, substituted aryloxyaryl,cyano, halogen, hydroxyl, nitro, oxo, thioxo, carboxyl, carboxyl esters,cycloalkyl, substituted cycloalkyl, thiol, alkylthio, substitutedalkylthio, arylthio, substituted arylthio, cycloalkylthio, substitutedcycloalkylthio, heteroarylthio, substituted heteroarylthio,heterocyclicthio, substituted heterocyclicthio, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy,substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,oxythiocarbonylamino, —OS(O)₂-alkyl, —OS(O)₂-substituted alkyl,—OS(O)₂-aryl, —OS(O)₂-substituted aryl, OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NR⁴⁰R⁴⁰ where each R⁴⁰ ishydrogen or alkyl, —NR⁴⁰S(O)₂-alkyl, —NR⁴⁰S(O)₂-substituted alkyl,—NR⁴⁰S(O)₂-aryl, —NR⁴⁰S(O)₂-substituted aryl, —NR⁴⁰S(O)₂-heteroaryl,—NR⁴⁰S(O)₂-substituted heteroaryl, —NR⁴⁰S(O)₂-heterocyclic,—NR⁴⁰S(O)₂-substituted heterocyclic, —NR⁴⁰S(O)₂—NR⁴⁰-alkyl,—NR⁴⁰S(O)₂—NR⁴⁰-substituted alkyl, —NR⁴⁰S(O)₂—NR⁴⁰-aryl,—NR⁴⁰S(O)₂—NR⁴⁰-substituted aryl, —NR⁴⁰S(O)₂—NR⁴⁰-heteroaryl,—NR⁴⁰S(O)₂—NR⁴⁰-substituted heteroaryl, —NR⁴⁰S(O)₂—NR⁴⁰-heterocyclic,and —NR⁴⁰S(O)₂—NR⁴⁰-substituted heterocyclic where each R⁴⁰ is hydrogenor alkyl.

“Alkoxy” refers to the group “alkyl-O—” which includes, by way ofexample, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy,sec-butoxy, n-pentoxy and the like.

“Substituted alkoxy” refers to the group “substituted alkyl-O—”.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—,substituted heteroaryl-C(O), heterocyclic-C(O)—, and substitutedheterocyclic-C(O)— provided that a nitrogen atom of the heterocyclic orsubstituted heterocyclic is not bound to the —C(O)— group wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic are as defined herein.

The term “aminoacyl” or as a prefix “carbamoyl” or “carboxamide” or“substituted carbamoyl” or “substituted carboxamide” refers to the group—C(O)NR⁴²R⁴² where each R⁴² is independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic and where each R⁴² is joined toform together with the nitrogen atom a heterocyclic or substitutedheterocyclic wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic are as defined herein.

“Acyloxy” refers to the groups alkyl-C(O)O—, substituted alkyl-C(O)O—,alkenyl-C(O)O—, substituted alkenyl-C(O)O—, alkynyl-C(O)O—, substitutedalkynyl-C(O)O—, aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—,substituted cycloalkyl-C(O)O—, heteroaryl-C(O)O—, substitutedheteroaryl-C(O)O—, heterocyclic-C(O)O—, and substitutedheterocyclic-C(O)O— wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Alkenyl” refers to alkenyl group preferably having from 2 to 6 carbonatoms and more preferably 2 to 4 carbon atoms and having at least 1 andpreferably from 1 to 2 sites of alkenyl unsaturation.

“Substituted alkenyl” refers to alkenyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl,aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl,carboxyl esters, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic.

“Alkynyl” refers to alkynyl group preferably having from 2 to 6 carbonatoms and more preferably 2 to 3 carbon atoms and having at least 1 andpreferably from 1-2 sites of alkynyl unsaturation.

“Substituted alkynyl” refers to alkynyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl,aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl,carboxyl esters, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic.

“Amino” refers to the group —NH₂.

“Substituted amino” refers to the group —NR⁴¹R⁴¹, where each R⁴¹ groupis independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-alkenyl,—SO₂-substituted alkenyl, —SO₂-cycloalkyl, —SO₂-substituted cycloalkyl,—SO₂-aryl, —SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substitutedheteroaryl, —SO₂-heterocyclic, —SO₂-substituted heterocyclic, providedthat both R⁴¹ groups are not hydrogen; or the R⁴¹ groups can be joinedtogether with the nitrogen atom to form a heterocyclic or substitutedheterocyclic ring.

“Acylamino” refers to the groups —NR⁴⁵C(O)alkyl, —NR⁴⁵C(O) substitutedalkyl, —NR⁴⁵C(O)cycloalkyl, —NR⁴⁵C(O) substituted cycloalkyl,—NR⁴⁵C(O)alkenyl, —NR⁴⁵C(O) substituted alkenyl, —NR⁴⁵C(O)alkynyl,—NR⁴⁵C(O) substituted alkynyl, —NR⁴⁵C(O)aryl, —NR⁴⁵C(O) substitutedaryl, —NR⁴⁵C(O)heteroaryl, —NR⁴⁵C(O) substituted heteroaryl,—NR⁴⁵C(O)heterocyclic, and —NR⁴⁵C(O) substituted heterocyclic where R⁴⁵is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are definedherein.

“Carbonyloxyamino” refers to the groups —NR⁴⁶C(O)O-alkyl,—NR⁴⁶C(O)O-substituted alkyl, —NR⁴⁶C(O)O-alkenyl, —NR⁴⁶C(O)O-substitutedalkenyl, —NR⁴⁶C(O)O-alkynyl, —NR⁴⁶C(O)O-substituted alkynyl,—NR⁴⁶C(O)β-cycloalkyl, —NR⁴⁶C(O)O-substituted cycloalkyl,—NR⁴⁶C(O)O-aryl, —NR⁴⁶C(O)O-substituted aryl, —NR⁴⁶C(O)O-heteroaryl,—NR⁴⁶C(O)O-substituted heteroaryl, —NR⁴⁶C(O)O-heterocyclic, and—NR⁴⁶C(O)O-substituted heterocyclic where R⁴⁶ is hydrogen or alkyl andwherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Aminocarbonyloxy” or as a prefix “carbamoyloxy” or “substitutedcarbamoyloxy” refers to the groups —OC(O)NR⁴⁷R⁴⁷ where each R⁴⁷ isindependently hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic, and substituted heterocyclic or where each R⁴⁷ is joinedto form, together with the nitrogen atom a heterocyclic or substitutedheterocyclic and wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic are as defined herein.

“Aminocarbonylamino” refers to the group —NR⁴⁹C(O)NR⁴⁹— where R⁴⁹ isselected from the group consisting of hydrogen and alkyl.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl) which condensed rings may ormay not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is the aryl group. Preferred aryls include phenyl andnaphthyl.

“Substituted aryl” refers to aryl groups, as defined herein, which aresubstituted with from 1 to 4, preferably 1-3, substituents selected fromthe group consisting of hydroxy, acyl, acylamino, carbonylaminothio,acyloxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, amidino, amino,substituted amino, aminoacyl, aminocarbonyloxy, aminocarbonylamino,aminothiocarbonylamino, aryl, substituted aryl, aryloxy, substitutedaryloxy, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy,substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,carboxyl, carboxyl esters cyano, thiol, alkylthio, substitutedalkylthio, arylthio, substituted arylthio, heteroarylthio, substitutedheteroarylthio, cycloalkylthio, substituted cycloalkylthio,heterocyclicthio, substituted heterocyclicthio, cycloalkyl, substitutedcycloalkyl, guanidino, halo, nitro, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic, oxycarbonylamino,oxythiocarbonylamino, —S(O)₂-alkyl, —S(O)₂-substituted alkyl,—S(O)₂-cycloalkyl, —S(O)₂-substituted cycloalkyl, —S(O)₂-alkenyl,—S(O)₂-substituted alkenyl, —S(O)₂-aryl, —S(O)₂-substituted aryl,—S(O)₂-heteroaryl, —S(O)₂-substituted heteroaryl, —S(O)₂-heterocyclic,—S(O)₂-substituted heterocyclic, —OS(O)₂-alkyl, —OS(O)₂-substitutedalkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NR⁵¹R⁵¹ where each R⁵¹ ishydrogen or alkyl, —NR⁵¹S(O)₂-alkyl, —NR⁵¹S(O)₂-substituted alkyl,—NR⁵¹S(O)₂-aryl, —NR⁵¹S(O)₂-substituted aryl, —NR⁵¹S(O)₂-heteroaryl,—NR⁵¹S(O)₂-substituted heteroaryl, —NR⁵¹S(O)₂-heterocyclic,—NR⁵¹S(O)₂-substituted heterocyclic, —NR⁵¹S(O)₂—NR⁵¹-alkyl,—NR⁵¹S(O)₂—NR⁵¹-substituted alkyl, —NR⁵¹S(O)₂—NR⁵¹-aryl,—NR⁵¹S(O)₂—NR⁵¹-substituted aryl, —NR⁵¹S(O)₂—NR⁵¹-heteroaryl,—NR⁵¹S(O)₂—NR⁵¹-substituted heteroaryl, —NR⁵¹S(O)₂—NR⁵¹-heterocyclic,—NR⁵¹S(O)₂—NR⁵¹-substituted heterocyclic where each R⁵¹ is hydrogen oralkyl, wherein each of the terms is as defined herein.

“Aryloxy” refers to the group aryl-O— that includes, by way of example,phenoxy, naphthoxy, and the like.

“Substituted aryloxy” refers to substituted aryl-O— groups.

“Aryloxyaryl” refers to the group -aryl-O-aryl.

“Substituted aryloxyaryl” refers to aryloxyaryl groups substituted withfrom 1 to 3 substituents on either or both aryl rings as defined abovefor substituted aryl.

“Carboxyl” refers to —COOH or salts thereof.

“Carboxyl esters” refers to the groups —C(O)O-alkyl, —C(O)O-substitutedalkyl, —C(O)O-aryl, and —C(O)O-substituted aryl wherein alkyl,substituted alkyl, aryl and substituted aryl are as defined herein.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including, by way of example,adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and thelike.

“Substituted cycloalkyl” refers to a cycloalkyl group, having from 1 to5 substituents selected from the group consisting of oxo (═O), thioxo(═S), alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aryl, substituted aryl, aryloxy,substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl, carboxylesters, cycloalkyl, substituted cycloalkyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic.

“Cycloalkoxy” refers to —O-cycloalkyl groups.

“Substituted cycloalkoxy” refers to —O-substituted cycloalkyl groups.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo andpreferably is fluoro or chloro.

“Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms,preferably from 1 to 10 carbon atoms, and 1 to 4 heteroatoms selectedfrom the group consisting of oxygen, nitrogen and sulfur within thering. Such heteroaryl groups can have a single ring (e.g., pyridinyl orfuryl) or multiple condensed rings (e.g., indolizinyl or benzothienyl).Preferred heteroaryls include pyridinyl, pyrrolyl, indolyl, thiophenyl,and furyl.

“Substituted heteroaryl” refers to heteroaryl groups that aresubstituted with from 1 to 3 substituents selected from the same groupof substituents defined for substituted aryl.

“Heteroaryloxy” refers to the group —O-heteroaryl and “substitutedheteroaryloxy” refers to the group —O-substituted heteroaryl.

“Heterocycle” or “heterocyclic” refers to a saturated or unsaturatedgroup having a single ring or multiple condensed rings, from 1 to 10carbon atoms and from 1 to 4 hetero atoms selected from the groupconsisting of nitrogen, sulfur or oxygen within the ring wherein, infused ring systems, one or more the rings can be aryl or heteroarylprovided that the point of attachment is at the heterocycle.

“Substituted heterocyclic” refers to heterocycle groups that aresubstituted with from 1 to 3 of the same substituents as defined forsubstituted cycloalkyl.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydro-isoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), piperidinyl, pyrrolidine, tetrahydrofuranyl, and thelike.

“Heterocyclyloxy” refers to the group —O-heterocyclic and “substitutedheterocyclyloxy” refers to the group —O-substituted heterocyclic.

“Thiol” or “mercapto” refers to the group —SH.

“Alkylsulfanyl” and “alkylthio” refer to the groups —S-alkyl where alkylis as defined above.

“Substituted alkylthio” and “substituted alkylsulfanyl” refer to thegroup —S-substituted alkyl is as defined above.

“Cycloalkylthio” or “cycloalkylsulfanyl” refers to the groups—S-cycloalkyl where cycloalkyl is as defined above.

“Substituted cycloalkylthio” refers to the group —S-substitutedcycloalkyl where substituted cycloalkyl is as defined above.

“Arylthio” refers to the group —S-aryl and “substituted arylthio” refersto the group —S-substituted aryl where aryl and substituted aryl are asdefined above.

“Heteroarylthio” refers to the group —S-heteroaryl and “substitutedheteroarylthio” refers to the group —S-substituted heteroaryl whereheteroaryl and substituted heteroaryl are as defined above.

“Heterocyclicthio” refers to the group —S-heterocyclic and “substitutedheterocyclicthio” refers to the group —S-substituted heterocyclic whereheterocyclic and substituted heterocyclic are as defined above.

The term “amino acid” refers to any of the naturally occurring aminoacids, as well as synthetic analogs (e.g., D-stereoisomers of thenaturally occurring amino acids, such as D-threonine) and derivativesthereof. α-Amino acids comprise a carbon atom to which is bonded anamino group, a carboxyl group, a hydrogen atom, and a distinctive groupreferred to as a “side chain”. The side chains of naturally occurringamino acids are well known in the art and include, for example, hydrogen(e.g., as in glycine), alkyl (e.g., as in alanine, valine, leucine,isoleucine, proline), substituted alkyl (e.g., as in threonine, serine,methionine, cysteine, aspartic acid, asparagine, glutamic acid,glutamine, arginine, and lysine), arylalkyl (e.g., as in phenylalanineand tryptophan), substituted arylalkyl (e.g., as in tyrosine), andheteroarylalkyl (e.g., as in histidine). Unnatural amino acids are alsoknown in the art, as set forth in, for example, Williams (ed.),Synthesis of Optically Active .alpha.-Amino Acids, Pergamon Press(1989); Evans et al., J. Amer. Chem. Soc., 112:4011-4030 (1990); Pu etal., J. Amer. Chem. Soc., 56:1280-1283 (1991); Williams et al., J. Amer.Chem. Soc., 113:9276-9286 (1991); and all references cited therein. Thepresent invention includes the side chains of unnatural amino acids aswell.

“Pharmaceutically acceptable salt” refers to pharmaceutically acceptablesalts of a compound, which salts are derived from a variety of organicand inorganic counter ions well known in the art and include, by way ofexample only, sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium, and the like; and when the molecule contains a basicfunctionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,oxalate and the like.

The term “prodrug” refers to compounds of this invention which have beenmodified to include a physiologically and biocompatible removable groupwhich group is removed in vivo to provide for the active drug, apharmaceutically acceptable salt thereof or a biologically activemetabolite thereof. Suitable removable groups are well known in the artand particularly preferred removable groups include esters of thecarboxylic acid moiety on the glycine substituent. Preferably suchesters include those derived from alkyl alcohols, substituted alkylalcohols, hydroxy substituted aryls and heteroaryls and the like.Another preferred removable group are the amides formed from thecarboxylic acid moiety on the glycine substituent. Suitable amides arederived from amines of the formula HNR²⁰R²¹ where R²⁰ and R²¹ areindependently hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, and the like.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,etc.) are not intended for inclusion herein. In such cases, the maximumnumber of such substituents is three. That is to say that each of theabove definitions is constrained by a limitation that, for example,substituted aryl groups are limited to—substituted aryl-(substitutedaryl)-substituted aryl.

Similarly, it is understood that the above definitions are not intendedto include impermissible substitution patterns (e.g., methyl substitutedwith 5 fluoro groups or a hydroxyl group alpha to ethenylic oracetylenic unsaturation). Such impermissible substitution patterns arewell known to the skilled artisan.

The Methods of the Invention

The present invention provides methods of modulating HIF and/or EPO byinhibiting HIFα hydroxylation, thereby stabilizing HIF and activatingHIF-regulated gene expression. The methods can be applied to theprevention, pretreatment, or treatment of conditions associated with HIFand or EPO including anemic, ischemic and hypoxic conditions.

Treatment of HIF-Associated Conditions

Ischemia and Hypoxia are two conditions associated with HIF and include,but are not limited to, myocardial infarction, liver ischemia, renalischemia, and stroke; peripheral vascular disorders, ulcers, burns, andchronic wounds; pulmonary embolism; and ischemic-reperfusion injury,including, for example, ischemic-reperfusion injury associated withsurgery and organ transplantation. In one embodiment, the presentinvention provides methods of stabilizing HIFα before, during, orimmediately after ischemia or hypoxia, particularly in association withmyocardial infarction, stroke, or renal ischemic-reperfusion injury.

In one aspect, the invention provides methods for treating variousischemic and hypoxic conditions, in particular, using the compoundsdescribed herein. In one embodiment, the methods of the inventionproduce therapeutic benefit when administered following ischemia orhypoxia. For example, the methods of the invention produce a dramaticdecrease in morbidity and mortality following myocardial infarction, anda significant improvement in heart architecture and performance.Further, the methods of the invention improve liver function whenadministered following hepatic toxic-ischemic injury. Hypoxia is asignificant component of liver disease, especially in chronic liverdisease associated with hepatotoxic compounds such as ethanol.Additionally, expression of genes known to be induced by HIFα, e.g.,nitric oxide synthase and glucose transporter-1, is increased inalcoholic liver disease. (See, e.g., Areel et al. (1997) Hepatology25:920-926; Strubelt (1984) Fundam. Appl. Toxicol. 4:144-151; Sato(1983) Pharmacol Biochem Behav 18 (Suppl. 1):443-447; Nanji et al.(1995) Am. J. Pathol. 146:329-334; and Morio et al. (2001) Toxicol.Appl. Pharmacol. 172:44-51.)

Therefore, the present invention provides methods of treating conditionsassociated with ischemia or hypoxia, the method comprising administeringa therapeutically effective amount of a compound or a pharmaceuticallyacceptable salt thereof, alone or in combination with a pharmaceuticallyacceptable excipient, to a subject. In one embodiment, the compound isadministered immediately following a condition producing acute ischemia,e.g., myocardial infarction, pulmonary embolism, intestinal infarction,ischemic stroke, and renal ischemic-reperfusion injury. In anotherembodiment, the compound is administered to a patient diagnosed with acondition associated with the development of chronic ischemia, e.g.,cardiac cirrhosis, macular degeneration, pulmonary embolism, acuterespiratory failure, neonatal respiratory distress syndrome, andcongestive heart failure. In yet another embodiment, the compound isadministered immediately after a trauma or injury.

In another aspect, the invention provides methods for treating a patientat risk of developing an ischemic or hypoxic condition, e.g.,individuals at high risk for atherosclerosis, etc., using the compoundsdescribed herein. Risk factors for atherosclerosis include, e.g.,hyperlipidemia, cigarette smoking, hypertension, diabetes mellitus,hyperinsulinemia, and abdominal obesity. Therefore, the presentinvention provides methods of preventing ischemic tissue injury, themethod comprising administering a therapeutically effective amount of acompound or a pharmaceutically acceptable salt thereof, alone or incombination with a pharmaceutically acceptable excipient, to a patientin need. In one embodiment, the compound can be administered based onpredisposing conditions, e.g., hypertension, diabetes, occlusivearterial disease, chronic venous insufficiency, Raynaud's disease,chronic skin ulcers, cirrhosis, congestive heart failure, and systemicsclerosis.

In one specific embodiment, the methods are used to increasevascularization and/or granulation tissue formation in damaged tissue,wounds, and ulcers. For example, compounds of the invention have beenshown to be effective in stimulating granulation tissue formation inwound healing. Granulation tissue contains newly formed, leaky bloodvessels and a provisional stroma of plasma proteins, such as fibrinogenand plasma fibronectin. Release of growth factors from inflammatorycells, platelets, and activated endothelium, stimulates fibroblast andendothelial cell migration and proliferation within the granulationtissue. Ulceration can occur if vascularization or neuronal stimulationis impaired. The methods of the invention are effective at promotinggranulation tissue formation. Thus, the invention provides methods fortreating a patient having tissue damage due to, e.g., an infarct, havingwounds induced by, e.g., trauma or injury, or having chronic wounds orulcers produced as a consequence of a disorder, e.g., diabetes. Themethod comprises administering a therapeutically effective amount of acompound or a pharmaceutically acceptable salt thereof, alone or incombination with a pharmaceutically acceptable excipient, to a patientin need.

In another aspect, the invention provides methods of using the compoundsto pretreat a subject to decrease or prevent the development of tissuedamage associated with ischemia or hypoxia. The methods of the inventionproduce therapeutic benefit when administered immediately before acondition involving ischemia or hypoxia. For example, application of themethods of the invention prior to induction of myocardial infarctionshows statistically significant improvement in heart architecture andperformance. Further, the methods of the invention produce therapeuticbenefit when administered immediately before and duringischemic-reperfusion injury, significantly reducing diagnosticparameters associated with renal failure.

Therefore, the invention provides methods of pretreating a subject todecrease or prevent the tissue damage associated with ischemia orhypoxia, the method comprising administering a therapeutically effectiveamount of a compound or a pharmaceutically acceptable salt thereof,alone or in combination with a pharmaceutically acceptable excipient, toa patient with a history of ischemic disorders, e.g., myocardialinfarctions, or having symptoms of impending ischemia, e.g., anginapectoris. In another embodiment, the compound can be administered basedon physical parameters implicating possible ischemia, e.g., individualsplaced under general anesthesia or temporarily working at highaltitudes. In yet another embodiment, the compounds may be used in organtransplants to pretreat organ donors and to maintain organs removed fromthe body prior to implantation in the recipient.

Previous studies have shown that certain compounds used in the methodsof the present invention are effective inhibitors of procollagen prolyl4-hydroxylase. While it is recognized that recovery from an initialinfarct or wound requires connective tissue deposition within thenecrotic region, the present invention demonstrates no adverse affectsof treatment with respect to scar formation. Thus, based on the benefitsprovided by certain compounds of the invention on treatment andprevention of hypoxic tissue damage and fibrosis, the present inventioncontemplates a “dual-therapy” approach to treatment or prevention ofconditions involving ischemia or hypoxia, including ischemia or hypoxiaassociated with subsequent reactive fibrosis, e.g., myocardialinfarction and resultant congestive heart failure. The method may useone compound that inhibits more than one 2-oxoglutarate dioxygenaseenzyme, e.g., HIF prolyl hydroxylase and procollagen prolyl4-hydroxylase, with either the same specificity or with differentspecificities. Alternatively, the method may use a combination ofcompounds wherein each compound specifically inhibits only one2-oxoglutarate dioxygenase enzyme, e.g., one compound specificallyinhibits HIF prolyl hydroxylase and a second compound specificallyinhibits procollagen prolyl 4-hydroxylase.

In one aspect, a compound of the invention inhibits one or more2-oxoglutarate dioxygenase enzymes. In one embodiment, the compoundinhibits at least two 2-oxoglutarate dioxygenase family members, e.g.,HIF prolyl hydroxylase and HIF asparagine-hydroxylase (FIH-1), witheither the same specificity or with differential specificity. In anotherembodiment, the compound is specific for one 2-oxoglutarate dioxygenase,e.g., HIF prolyl hydroxylase, and shows little to no specificity forother family members.

The compounds can be administered in combination with various othertherapeutic approaches. In one embodiment, the compound is administeredwith another 2-oxoglutarate dioxygenase inhibitor, wherein the twocompounds have differential specificity for individual 2-oxoglutaratedioxygenase family members. The two compounds may be administered at thesame time as a ratio of one relative to the other. Determination of aratio appropriate to a given course of treatment or a particular subjectis within the level of skill in the art. Alternatively, the twocompounds may be administered consecutively during a treatment timecourse, e.g., following myocardial infarction. In a particularembodiment, one compound specifically inhibits HIF prolyl hydroxylaseenzyme activity, and a second compound specifically inhibits procollagenprolyl 4-hydroxylase enzyme activity. In another specific embodiment,one compound specifically inhibits HIF prolyl hydroxylase enzymeactivity, and a second compound specifically inhibits HIFasparaginyl-hydroxylase enzyme activity. In another embodiment, thecompound is administered with another therapeutic agent having adifferent mode of action, e.g., an ACE inhibitor (ACEI), angiotensin-IIreceptor blocker (ARB), statin, diuretic, digoxin, carnitine, etc.

Treatment EPO-Associated Conditions

The present invention provides methods of increasing endogenouserythropoietin (EPO). These methods can be applied in vivo, e.g., inblood plasma, or in vitro, e.g., in cell culture conditioned media. Theinvention further provides methods of increasing endogenous EPO levelsto prevent, pretreat, or treat EPO-associated conditions, including,e.g., conditions associated with anemia and neurological disorders.Conditions associated with anemia include disorders such as acute orchronic kidney disease, diabetes, cancer, ulcers, infection with virus,e.g., HIV, bacteria, or parasites; inflammation, etc. Anemic conditionscan further include those associated with procedures or treatmentsincluding, e.g., radiation therapy, chemotherapy, dialysis, and surgery.Disorders associated with anemia additionally include abnormalhemoglobin and/or erythrocytes, such as found in disorders such asmicrocytic anemia, hypochromic anemia, aplastic anemia, etc.

The present methods can be used to increase endogenous EPO in a subjectundergoing a specific treatment or procedure, prophylactically orconcurrently, for example, an HIV-infected anemic patient being treatedwith azidothymidine (zidovudine) or other reverse transcriptaseinhibitors, an anemic cancer patient receiving cyclic cisplatin- ornon-cisplatin-containing chemotherapeutics, or an anemic or non-anemicpatient scheduled to undergo surgery. Methods of increasing endogenousEPO can also be used to prevent, pretreat, or treat EPO-associatedconditions associated with nerve damage or neural tissue degenerationincluding, but not limited to, stroke, trauma, epilepsy, spinal cordinjury, and neurodegenerative disorders.

Additionally, the methods can be used to increase endogenous EPO levelsin an anemic or non-anemic patient scheduled to undergo surgery toreduce the need for allogenic blood transfusions or to facilitatebanking of blood prior to surgery. The small decreases in hematocritthat typically occur after presurgical autologous blood donation do notstimulate an increase in endogenous EPO or in compensatoryerythropoiesis. However, preoperative stimulation of endogenous EPOwould effectively increase erythrocyte mass and autologous donationvolumes while maintaining higher hematocrit levels, and such methods arespecifically contemplated herein. In some surgical populations,particularly those individuals who experience surgical blood losses inexcess of 2 liters, the methods of the invention could be applied toreduce allogeneic blood exposure. Crosby (2002) Amer. J. Therap.9:371-376.

The methods of the invention can also be used to enhance athleticperformance, improve exercise capacity, and facilitate or enhanceaerobic conditioning. Such methods can be used, e.g., by athletes tofacilitate training and by soldiers to improve, e.g., stamina andendurance.

The methods of the invention have been shown to increase endogenouserythropoietin levels in media from cultured cells treated in vitro andin blood plasma from animals treated in vivo. Although the kidney is themajor source of erythropoietin in the body, other organs, includingbrain, liver, and bone marrow, can and do synthesize erythropoietin uponappropriate stimulation. Using the methods of the invention, endogenouserythropoietin expression can be increased in various organs of thebody, including brain, kidney, and liver. Indeed, methods of theinvention even increase endogenous erythropoietin levels in animals thathave undergone bilateral nephrectomy.

The methods of the invention demonstrate that erythropoietin levels canbe increased even when kidney function is compromised. Although theinvention is not to be limited by the mechanism by which erythropoietinis produced, the decrease in erythropoietin secretion typically seenduring kidney failure may be due to hyperoxia in renal tissue due toincreased flowthrough/reperfusion. Priyadarshi et al. (2002) Kidney Int.61:542-546.

Further, the methods of the invention increase the hematocrit and bloodhemoglobin level in animals treated in vivo. The increases in plasmaEPO, hematocrit, and blood hemoglobin in response to the compounds usedin the methods of the invention are dose-sensitive; however, dosingregimes can be established which produce a constant, controlled level ofresponse to the compounds of the invention. Further, treatment withcompounds of the invention can correct anemia, for example, induced by atoxic compound such as the chemotherapeutic agent cisplatin, or due toblood loss, e.g., trauma, injury, parasites, or surgery.

The increase in hematocrit and blood hemoglobin in animals treated withcompounds of the invention is preceded by an increase in the percentageof circulating immature red blood cells (reticulocytes) within theblood. As such, the invention contemplates the use of the compounds ofthe invention in methods to increase reticulocyte levels in the blood ofanimals for production of cell-free reticulocyte lysates as describedby, e.g., Pelham and Jackson. Eur. J. Biochem. 67:247-256 (1976).Circulating reticulocyte levels are increased in animals, e.g., rabbits,etc., by treatment with compounds of the invention, alone or incombination with another compound such as, e.g., acetylphenylhydrazine,etc. The blood is collected, and reticulocytes are pelleted bycentrifugation and lysed with distilled water. Extracts can be furtherprocessed using any appropriate methodology known to those skilled inthe art. See, e.g., Jackson and Hunt (1983) Methods Enzymol. 96:50-74.

The compounds of this invention can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Second Edition, Wiley, New York, 1991, and references citedtherein.

Furthermore, the compounds of this invention will typically contain oneor more chiral centers. Accordingly, if desired, such compounds can beprepared or isolated as pure stereoisomers, i.e., as individualenantiomers or diastereomers, or as stereoisomer-enriched mixtures. Allsuch stereoisomers (and enriched mixtures) are included within the scopeof this invention, unless otherwise indicated. Pure stereoisomers (orenriched mixtures) may be prepared using, for example, optically activestarting materials or stereoselective reagents well-known in the art.Alternatively, racemic mixtures of such compounds can be separatedusing, for example, chiral column chromatography, chiral resolvingagents and the like.

The compounds of this invention are preferably prepared by a convergentsynthetic protocol combining the amino entity and the substitutedisoquinoline acetic acid derivative under conventional couplingconditions as illustrated in Scheme 1 below:

Specifically, in Scheme 1, an appropriately substituted 3-protectedcarboxyl isoquinoline, compound 1, is combined with at least astoichiometric amount and preferably an excess of the substituted amineor the N-alkyl derivative thereof, compound 2. The reaction is conductedunder conventional coupling conditions well known in the art. In oneembodiment, the reaction is conducted in the presence of sodiummethoxide in methanol under elevated reaction temperatures andpreferably at reflux. The reaction is continued until it issubstantially complete which typically occurs within about 1 to 48hours. Upon reaction completion, compound 3, can be recovered byconventional techniques such as neutralization, extraction,precipitation, chromatography, filtration and the like; or,alternatively, used in the next step without purification and/orisolation.

Alternatively, coupling of the substituted 3-protected carboxylisoquinoline, compound 1, is combined with the substituted amine or theN-alkyl derivative thereof, compound 2, can proceed via conventionalpeptide coupling procedures well known in the art. This couplingreaction is typically conducted using well-known coupling reagents suchas carbodiimides, BOP reagent(benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphonate) and the like. Suitable carbodiimides include, byway of example, dicyclohexylcarbodiimide (DCC),1-(3-dimethylamino-propyl)-3-ethylcarbodiimide (DECI) and the like. Ifdesired, polymer supported forms of carbodiimide coupling reagents mayalso be used including, for example, those described in TetrahedronLetters, 34(48), 7685 (1993). Additionally, well-known couplingpromoters, such as N-hydroxysuccinimide, 1-hydroxybenzotriazole and thelike, may be used to facilitate the coupling reaction.

This coupling reaction is typically conducted by contacting compound 1(typically as the free acid) with about 1 to about 2 equivalents of thecoupling reagent and at least one equivalent, preferably about 1 toabout 1.2 equivalents, of compound 2, in an inert diluent, such asdichloromethane, chloroform, acetonitrile, tetrahydrofuran,N,N-dimethylformamide and the like. Generally, this reaction isconducted at a temperature ranging from about 0° C. to about 37° C. forabout 12 to about 24 hours. Upon completion of the reaction, compound 3is recovered by conventional methods including neutralization,extraction, precipitation, chromatography, filtration, and the like.

Alternatively, the substituted 3-protected carboxyl isoquinoline,compound 1, can be converted into an acid halide and the acid halidecoupled with compound 2 to provide for compound 3. The acid halide ofcompound 1 can be prepared by contacting compound 1 with an inorganicacid halide, such as thionyl chloride, phosphorous trichloride,phosphorous tribromide or phosphorous penta-chloride, or preferably,with oxalyl chloride under conventional conditions. Generally, thisreaction is conducted using about 1 to 5 molar equivalents of theinorganic acid halide or oxalyl chloride, either neat or in an inertsolvent, such as dichloromethane or carbon tetrachloride, at temperaturein the range of about 0° C. to about 80° C. for about 1 to about 48hours. A catalyst, such as DMF, may also be used in this reaction.

The acid halide (not shown) is then contacted with at least oneequivalent, preferably about 1.1 to about 1.5 equivalents, of compound2, in an inert diluent, such as dichloromethane, at a temperatureranging from about −70° C. to about 40° C. for about 1 to about 24hours. Preferably, this reaction is conducted in the presence of asuitable base to scavenge the acid generated during the reaction.Suitable bases include, by way of example, tertiary amines, such astriethylamine, diisopropylethylamine, N-methyl-morpholine and the like.Alternatively, the reaction can be conducted under Schotten-Baumann-typeconditions using aqueous alkali, such as sodium hydroxide and the like.Upon completion of the reaction, compound 3 is recovered by conventionalmethods including neutralization, extraction, precipitation,chromatography, filtration, and the like.

In one embodiment, the nitrogen atom of the isoquinoline ring system canbe oxidized via conventional techniques to provide for the correspondingN-oxide compound, compounds 4 and 5. Oxidation can proceed by use ofconventional oxidizing agents such as m-chloroperbenzoic acid orhydrogen peroxide under conventional conditions. As depicted in Scheme1, N-oxide formation can occur either with the substituted 3-protectedcarboxyl isoquinoline, compound 1, or with compound 3.

The starting materials for use in the reactions found in Scheme 1 areeither commercially available or can be prepared by methods well knownin the art. For example, glycine and N-alkylglycines such as sarcosine,N-ethylglycine, and the like are commercially available from AldrichChemical Company, Milwaukee, Wis., USA. (“Aldrich”).

The synthesis of substituted isoquinoline acetic acids are also wellknown in the art and are described in detail by, for example, Weidmann,et al., U.S. Pat. No. 6,093,730 which is incorporated herein byreference in its entirety. One particular method for preparation of suchderivatives are set forth in Scheme 2 below:

Specifically, in Scheme 2, commercially available4-phenylsulfanyl-phthalonitrile, compound 6, is hydrolyzed to thecorresponding diacid, compound 7, under conventional conditions such astreatment with a 1:1 mixture of 50% aqueous KOH/methanol. The reactionis continued until it is substantially complete which typically occurswithin about 48 to 96 hours. Upon reaction completion, the resultingdiacid, compound 7, can be recovered by conventional techniques such asneutralization, extraction, precipitation, chromatography, filtrationand the like; or, alternatively, used in the next step withoutpurification and/or isolation.

Compound 7 is cyclized in the presence of a stoichiometric equivalent ofglycine. The reaction is conducted in the solid phase by first forming ahomogeneous mixture of the reagents and then heating the mixture to anelevated temperature to form a molten mass. Preferably, the reaction isheated to over 200° C. and more preferably from about 210° to about 220°C. The reaction is continued until it is substantially complete whichtypically occurs within about 48 to 96 hours. Upon reaction completion,the resulting phthalimide, compound 8, can be recovered by conventionaltechniques such as neutralization, extraction, precipitation,chromatography, filtration and the like; or, alternatively, used in thenext step without purification and/or isolation.

Conventional esterification of compound 8 leads to compound 9 where R⁸is alkyl. This compound is then subject to ring expansion under basicconditions. Specifically, compound 9 is contacted with an stoichiometricexcess, preferably 2 equivalents, of sodium or potassium alkoxide, suchas sodium butoxide, in a suitable solvent such as n-butanol andmaintained at an elevated temperature of from about 70° C. to about 120°C. and preferably from about 95° C. to about 100° C. The reaction iscontinued until it is substantially complete which typically occurswithin about 0.5 to 6 hours. Upon reaction completion, the resultingisoquinoline isomers, compounds 9 and 10 can be recovered byconventional techniques such as neutralization, extraction,precipitation, chromatography, filtration and the like; or,alternatively, used in the next step without purification and/orisolation.

The reaction conditions set forth above can lead to transesterificationof the ester functionality (if R⁸ is not n-butyl). In any event, thealkyl moiety of the ester group serves as a suitable protecting groupfor the carboxyl functionality on compound 9 and is depicted as Pg¹ incompound 1 of Scheme 1.

As is apparent, the hydroxy functionality at the 1 position is subjectto numerous derivation schemes that are well known in the art. Suitablederivations include formation of alkoxy, substituted alkoxy, aryloxy,substituted aryloxy, heteroaryloxy, substituted heteroaryloxy,heterocycyloxy, substituted heterocycloxy, halogenation, dehalogenation(to provide for hydrogen at this position), alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl products.Still further, the hydroxyl group can be modified using art recognizedprocedures to provide for —N(R⁷)R⁶ derivatives which can be achieved byreacting the halo substituent with a suitable amine. Similarly, sulfanyland oxidized sulfanyl derivatives can be prepared by conventionalmethods such as reacting the hydroxyl group with phosphorouspentasulfide, Lawesson's reagent, or the like, optionally followed byreaction of the resulting sulfhydryl group with an alkylating agents,such as ethyl iodide or the like, to give an alkylsulfanyl derivative.Sulfanyl derivatives may further be oxidized with standard peroxy acidreagents, such as m-chloroperbenzoic acid.

Still further, substitution on the phenyl ring of the isoquinolinecompounds is achieved by appropriate choice of starting materials. Manyof these starting materials are commercially available such as4-phenoxy-phthalonitrile (Aldrich), and the like. Alternatively,compounds such as 4-(2,6-dimethylphenoxy)-phthalonitrile can be preparedby art-recognized techniques.

Alternatively, commercially available substituted phthalic anhydride orphthalic acid can be used in place of compound 7 in Scheme 1. Suchanhydrides include, for example, 3-fluorophthalic anhydride (Aldrich),3-nitrophthalic anhydride (Aldrich), 3-chlorophthalic anhydride (TCIAmerica, Portland Oreg. 97203 “TCI”) and the like. Such acids include,for example, 4-trifluoromethyl-phthalic acid (TCI) and the like.

Testing and Administration

Biological Testing

The biological activity of the compounds of the invention may beassessed using any conventionally known methods. Suitable assay methodsare well known in the art. The following assays are presented only asexamples and are not intended to be limiting. The compounds of theinvention are active in at least one of the following assays.

Cell-Based HIFα Stabilization Assay

Human cells derived from various tissues were separately seeded into 35mm culture dishes and grown at 37° C., 20% O₂, 5% CO₂ in standardculture medium, e.g., DMEM, 10% FBS. When cell layers reachedconfluence, the media was replaced with OPTI-MEM media (Invitrogen LifeTechnologies, Carlsbad Calif.) and cell layers were incubated forapproximately 24 hours in 20% O₂, 5% CO₂ at 37° C. Compound or 0.013%DMSO was then added to existing medium, and incubation was continuedovernight.

Following incubation, the media was removed, centrifuged, and stored foranalysis (see VEGF and EPO assays below). The cells were washed twotimes in cold phosphate buffered saline (PBS) and then lysed in 1 ml of10 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 0.5% IGEPAL (Sigma-Aldrich,St. Louis Mo.), and a protease inhibitor mix (Roche MolecularBiochemicals) for 15 minutes on ice. Celllysates were centrifuged at3,000×g for 5 minutes at 4° C., and the cytosolic fractions(supernatant) were collected. The nuclei (pellet) were resuspended andlysed in 100 μl of 20 mM HEPES (pH 7.2), 400 mM NaCl, 1 mM EDTA, 1 mMdithiothreitol, and a protease mix (Roche Molecular Biochemicals),centrifuged at 13,000×g for 5 minutes at 4° C., and the nuclear proteinfractions (supernatant) were collected.

Nuclear fractions were analyzed for HIF-1α using a QUANTIKINEimmunoassay (R&D Systems, Inc., Minneapolis Minn.) according to themanufacturer's instructions.

Cell-Based VEGF and EPO ELISA Assays

Conditioned media collected from cell cultures as described above wasanalyzed for vascular endothelial growth factor (VEGF) and/orerythropoietin (EPO) expression using an appropriate QUANTIKINEimmunoassay (R&D Systems) according to the manufacturer's instructions.

Oxygen Consumption Assay

Oxygen Sensor cell culture plates (BD Biosciences) contain a rutheniumcomplex which is more fluorescent in the absence of oxygen. Therefore,the fluorescent read-out is increased by the presence ofoxygen-consuming cells in the plate, which change the equilibrium tolower oxygen saturation and higher fluorescence. A compound thatstabilizes HIF by inhibiting hydroxylation is expected to decreaseoxygen consumption by decreasing oxygen consumed by the hydroxylationevent itself and/or by shifting cellular metabolism from aerobic toanaerobic energy production.

Human cells derived from adenovirus-transformed fetal kidney epithelium(293A) or cervical epithelial adenocarcinoma (HeLa) (American TypeCulture Collection, Manassas Va.) were grown to confluence in media(high glucose DMEM (Mediatech, Inc., Herndon Va.), 1%penicillin/streptomycin mixture (Mediatech), 1% fetal bovine serum) at37° C., 10% CO₂. Cells were collected and resuspended in media at adensity of 500,000 cells/ml. The cell suspension was distributed at 0.2ml/well into each well of an Oxygen Biosensor 96-well cell culture plate(BD Biosciences, Bedford Mass.). The following treatments were added in10 μl volumes to triplicate sets of wells: (1) 0.5% DMSO; (2) 200 μMsodium dodecyl sulfate; or (3) 1, 10, or 50 μM compound.

Cultures were incubated at 37° C., 10% CO₂ for 72 hours and plates werethen read in an FL600 flourimeter (Biotek Instruments, Inc., WinooskiVt.) at an excitation wavelength of 485 nm and emission wavelength of590 nm. Data was plotted as a function of fold change relative to DMSOcontrol (O₂ consumption) or absorbance at a wavelength of 450 nm (WST-1)and descriptive statistical analysis was performed using EXCEL software(Microsoft Corporation, Bellevue Wash.).

HIF-PH2 (PHD2) Assay

Material

HIF-PH2 (EGLN1) was expressed from Hi5 cells and partially purifiedthrough a SP ion exchange chromatography column. Ketoglutaric acid-[1-14C]-sodium salt was obtained from Perkin-Elmer. Alphaketoglutaricacid sodium salt was purchased from SIGMA. HPLC purified DLD19 Peptide(Acetyl-DLDLEMLAPYIPMDDDFQL-CONH2)(SEQ ID NO:4) was made by Synpep.

HIF-PH2 (EGLN1) was expressed from insect Hi5 cells and partiallypurified through a SP ion exchange chromatography column. Enzymeactivity was determined by capturing ¹⁴CO₂ using an assay described byKivirikko and Myllyla (1982, Methods Enzymol 82:245-304). Assayreactions contained 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acidsodium salt, 0.30 μCi/mlketoglutaric acid μ-[1-¹⁴C]-sodium salt; PerkinElmer, Wellesley Mass.), 40 μM FeSO₄, 1 mM ascorbate, 1541.8 units/mlCatalase, with or without 50 μM peptide substrate(Acetyl-DLDLEMLAPYIPMDDDFQL-CONH₂)(SEQ ID NO:4) and variousconcentrations of compound of the invention. Reactions were initiated byaddition of HIF-PH2 enzyme.

The peptide-dependent percent turnover was calculated by subtractingpercent turnover in the absence of peptide from percent turnover in thepresence of substrate peptide. Percent inhibition and IC₅₀ werecalculated using peptide-dependent percent turnover at given inhibitorconcentrations. Calculation of IC₅₀ values for each inhibitor wasconducted using GraFit software (Erithacus Software Ltd., Surrey UK).

Pharmaceutical Formulations and Routes of Administration

The compositions of the present invention can be delivered directly orin pharmaceutical compositions along with suitable carriers orexcipients, as is well known in the art. Present methods of treatmentcan comprise administration of an effective amount of a compound of theinvention to a subject having or at risk for anemia due to, e.g.,chronic renal failure, diabetes, cancer, AIDS, radiation therapy,chemotherapy, kidney dialysis, or surgery. In a preferred embodiment,the subject is a mammalian subject, and in a most preferred embodiment,the subject is a human subject.

An effective amount of such agents can readily be determined by routineexperimentation, as can the most effective and convenient route ofadministration and the most appropriate formulation. Variousformulations and drug delivery systems are available in the art. See,e.g., Gennaro, A. R., ed. (1995) Remington's Pharmaceutical Sciences,supra.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, nasal, or intestinal administration and parenteraldelivery, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections. Theagent or composition thereof may be administered in a local rather thana systemic manner. For example, a suitable agent can be delivered viainjection or in a targeted drug delivery system, such as a depot orsustained release formulation.

The pharmaceutical compositions of the present invention may bemanufactured by any of the methods well-known in the art, such as byconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Asnoted above, the compositions of the present invention can include oneor more physiologically acceptable carriers such as excipients andauxiliaries that facilitate processing of active molecules intopreparations for pharmaceutical use.

Proper formulation is dependent upon the route of administration chosen.For injection, for example, the composition may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Fortransmucosal or nasal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. In a preferred embodiment of the presentinvention, the present compounds are prepared in a formulation intendedfor oral administration. For oral administration, the compounds can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject. The compoundsmay also be formulated in rectal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides.

Pharmaceutical preparations for oral use can be obtained as solidexcipients, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate. Also, wetting agents such as sodium dodecyl sulfate may beincluded.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations for oral administration include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

In one embodiment, the compounds of the present invention can beadministered transdermally, such as through a skin patch, or topically.In one aspect, the transdermal or topical formulations of the presentinvention can additionally comprise one or multiple penetrationenhancers or other effectors, including agents that enhance migration ofthe delivered compound. Transdermal or topical administration could bepreferred, for example, in situations in which location specificdelivery is desired.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orany other suitable gas. In the case of a pressurized aerosol, theappropriate dosage unit may be determined by providing a valve todeliver a metered amount. Capsules and cartridges of, for example,gelatin, for use in an inhaler or insufflator may be formulated. Thesetypically contain a powder mix of the compound and a suitable powderbase such as lactose or starch.

Compositions formulated for parenteral administration by injection,e.g., by bolus injection or continuous infusion can be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Formulations for parenteral administration include aqueoussolutions or other compositions in water-soluble form.

Suspensions of the active compounds may also be prepared as appropriateoily injection suspensions. Suitable lipophilic solvents or vehiclesinclude fatty oils such as sesame oil and synthetic fatty acid esters,such as ethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

As mentioned above, the compositions of the present invention may alsobe formulated as a depot preparation. Such long acting formulations maybe administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thepresent compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Suitable carriers for the hydrophobic molecules of the invention arewell known in the art and include co-solvent systems comprising, forexample, benzyl alcohol, a nonpolar surfactant, a water-miscible organicpolymer, and an aqueous phase. The co-solvent system may be the VPDco-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v ofthe nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol300, made up to volume in absolute ethanol. The VPD co-solvent system(VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in watersolution. This co-solvent system is effective in dissolving hydrophobiccompounds and produces low toxicity upon systemic administration.Naturally, the proportions of a co-solvent system may be variedconsiderably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied. For example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80, the fraction size of polyethyleneglycol may be varied, other biocompatible polymers may replacepolyethylene glycol, e.g., polyvinyl pyrrolidone, and other sugars orpolysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic molecules may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Liposomal delivery systemsare discussed above in the context of gene-delivery systems. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, thecompounds may be delivered using sustained-release systems, such assemi-permeable matrices of solid hydrophobic polymers containing theeffective amount of the composition to be administered. Varioussustained-release materials are established and available to those ofskill in the art. Sustained-release capsules may, depending on theirchemical nature, release the compounds for a few weeks up to over 100days. Depending on the chemical nature and the biological stability ofthe therapeutic reagent, additional strategies for protein stabilizationmay be employed.

For any composition used in the present methods of treatment, atherapeutically effective dose can be estimated initially using avariety of techniques well known in the art. For example, in a cellculture assay, a dose can be formulated in animal models to achieve acirculating concentration range that includes the IC₅₀ as determined incell culture. Dosage ranges appropriate for human subjects can bedetermined, for example, using data obtained from cell culture assaysand other animal studies.

A therapeutically effective dose of an agent refers to that amount ofthe agent that results in amelioration of symptoms or a prolongation ofsurvival in a subject. Toxicity and therapeutic efficacy of suchmolecules can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., by determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratio oftoxic to therapeutic effects is the therapeutic index, which can beexpressed as the ratio LD₅₀/ED₅₀. Agents that exhibit high therapeuticindices are preferred.

Dosages preferably fall within a range of circulating concentrationsthat includes the ED₅₀ with little or no toxicity. Dosages may varywithin this range depending upon the dosage form employed and the routeof administration utilized. The exact formulation, route ofadministration, and dosage should be chosen, according to methods knownin the art, in view of the specifics of a subject's condition.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety that are sufficient to modulateendogenous erythropoietin plasma levels as desired, i.e. minimaleffective concentration (MEC). The MEC will vary for each compound butcan be estimated from, for example, in vitro data. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. Agents or compositions thereof should be administeredusing a regimen which maintains plasma levels above the MEC for about10-90% of the duration of treatment, preferably about 30-90% of theduration of treatment, and most preferably between 50-90%. In cases oflocal administration or selective uptake, the effective localconcentration of the drug may not be related to plasma concentration.Alternatively, stimulation of endogenous erythropoietin may be achievedby 1) administering a loading dose followed by a maintenance dose, 2)administering an induction dose to rapidly achieve erythropoietin levelswithin a target range, followed by a lower maintenance dose to maintainhematocrit within a desired target range, or 3) repeated intermittentdosing.

The amount of agent or composition administered will, of course, bedependent on a variety of factors, including the sex, age, and weight ofthe subject being treated, the severity of the affliction, the manner ofadministration, and the judgment of the prescribing physician.

The present compositions may, if desired, be presented in a pack ordispenser device containing one or more unit dosage forms containing theactive ingredient. Such a pack or device may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.Compositions comprising a compound of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Suitable conditions indicated on the label may includetreatment of conditions, disorders, or diseases in which anemia is amajor indication.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein,and are specifically contemplated.

EXAMPLES

The invention is further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods that are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications fall within the scope of the appendedclaims.

Unless otherwise stated all temperatures are in degrees Celsius. Also,in these examples and elsewhere, abbreviations have the followingmeanings:

μl = microliter amu = atomic mass unit atm = atmosphere bs = broadsinglet ClCO₂iBu = isobutylchloro formate ClCONMe₂ = dimethylcarbamicchloride conc. = concentrated d = doublet DABCO =diazobicyclo[2.2.2]octane dd = doublet of doublets DMF = dimethylformamide DMSO = dimethyl sulfoxide Et₂SO₄ = ethyl sulfate EtI = ethyliodide EtOAc = ethyl acetate EtOH = ethanol EtOH = ethanol g = gram h =hour HATU = N-dimethylamino-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene-N-methylmethanaminium hexafluorophosphate N-oxideHBTU = 1-H-Benzotriazolium Hz = Hertz M = molar m = multiplet Me₂SO₄ =methyl sulfate Me₃OBF₄ = trimethylboroxine MeI = methyl iodide MeOCH₂I =iodomethoxy methane MeOH = methanol MeONa = sodium methoxide mg =milligram MHz = mega Hertz min = minute ml = milliliter mmol =millimolar N = normal NaOMe = sodium methoxide n-BuLi = n-butyl lithiumn-BuOH = n-butanol NEt₃ = triethyl amine PhCH₂Br = bromomethyl benzene q= quartet quint = quintuplet r.t. = room temperature R_(f) = retentionfactor s = second t = triplet TFA = trifluoro acetic acid THF =tetrahydrofuran TLC = thin layer chromatography wt % = weight percent

Example A-1(S)-2-[(6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid a.(S)-2-[6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid methyl ester

6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carboxylic-acid (can beobtained according to U.S. Pat. No. 6,093,730, October 1998, Weidmann etal.), 0.33 g, 0.5 ml of triethylamine, 0.38 g of HATU, and 0.151 g ofcommercial L-Alanine methyl ester hydrochloride were stirred in 15 mlCH₂Cl₂ at room temperature for 18 h to give, after silica gelchromatography (eluant 4:1 hexane-EtOAc). 0.220 g of(S)-2-[(6-benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid methyl ester as a white solid, MS-(+)-ion, M+1=415.8 amu.

b.(S)-2-[(6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid

0.200 g of the (S) methyl ester described in Example A-1 a) and 15 ml ofa 1.5 M solution of NaOH in methanol was stirred at room temperature for3 h and concentrated. The residue was dissolved in water and extractedwith EtOAc. The aqueous layer was acidified to pH ˜1 with hydrochloricacid and the resulting precipitate was collected by filtration, washedwith water, dried in a vacuum oven (70° C.) to give 0.174 g of(S)-2-[(6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid as an off-white solid, MS-(+)-ion, M+1=401.0 amu.

Example A-2(R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-hydroxy-propionicacid a. (1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid butyl ester

A mixture of 160 ml of butanol, 20.0 g of(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid (94.6 mmol) and 2.0 mlof concentrated sulfuric acid was refluxed with stirring for 24 h. Then5 g of sodium bicarbonate were added in portions, stirring continued atr.t. for 5 min and the solvent evaporated in vacuo. The residue waspartitioned between 100 ml of water and 100 ml of ethyl acetate. Theorganic phase was washed with 100 ml of brine, dried over sodium sulfateand was evaporated in vacuo to give a yellowish oil that latersolidified. 24.02 g of the title compound were obtained; MS-(+)-ion:M+1=261.9 amu.

b. 1,4-Dihydroxy-isoquinoline-3-carboxylic acid butyl ester

4.41 g of sodium (190 mmol) were dissolved in 250 ml of n-butanol withstirring. After the sodium was completely dissolved the solution wasallowed to cool to ambient temperature and a solution of 24.0 g (91.9mmol) of (1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid butyl esterin 150 ml of butanol was added with stirring. The solution was heated to100° C. within 30 min and stirred at this temperature for 1 h. Then themixture was allowed to cool to ambient temperature and was stored atambient temperature for 18 h. Then the pH of the mixture was adjusted to2 to 3 by the addition of aqueous 2N hydrochloric acid with stirring.Stirring was continued for 30 min before the solid component wasfiltered by suction. The filter cake was washed thoroughly with water,and dried in vacuo at 50° C. to give a white solid. 17.75 g of the titlecompound were obtained; MS-(+)-ion: M+1=262.1 amu.

c. 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

A mixture of 17.3 g (66.2 mmol) of1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester and 100 ml ofphosphorous oxychloride was stirred at ambient temperature for 1 h, andthen heated slowly with stirring in the course of 2 h to refluxtemperature. The mixture was refluxed gently with stirring for 30 min.After cooling to room temperature the excess phosphorous oxychloride wasevaporated in vacuo, and the residue was dissolved in 100 ml of ethylacetate. The solution was poured into 300 ml of a saturated aqueoussodium bicarbonate solution with stirring. The precipitate formed wasremoved by vacuum filtration. The organic phase was separated, and theaqueous phase was extracted with 3×100 ml of ethyl acetate. The combinedaqueous phases were dried over sodium sulfate, filtered through a pad ofsilica gel and evaporated in vacuo to give a brown oil that solidifiedlater. 11.37 g of the title compound were obtained; ¹H NMR (CDCl₃):δ=11.91 (s, 1H), 8.41 (m, 1H), 8.29 (m, 1H), 7.83 (m, 2H), 4.49 (t, 2H),1.84 (m, 2H), 1.48 (m, 2H), 0.99 (t, 3H).

d. 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid

A mixture of 9.23 g of 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (33 mmol), 90 ml of 2.5N aqueous sodium hydroxide solution,water (20 ml) and ethanol (110 ml) was refluxed with stirring for 2 h.Then the pH of the mixture was adjusted to 2 by the addition ofconcentrated aqueous hydrochloric acid. During the addition, thetemperature of the mixture was kept at 20° C. by cooling with an icebath. Stirring was then continued for 1 h before the solid component wasseparated by vacuum filtration. The filter cake was washed with waterand dried in vacuo at 85° C. to give a white powder. 6.64 g of the titlecompound were obtained; MS-(+)-ion: M+1=224.1 amu.

e.(R)-3-tert-Butoxy-2-[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid tert-butyl ester

To a mixture of 45 mg (0.2 mmol) of1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid, 76 mg (0.2 mmol) ofbenzotriazol-1-yl-(bis-dimethylamino-methylene)-oxonium hexafluorophosphate (HBTU), 50.8 mg (R)-2-amino-3-tert-butoxy-propionic acidtert-butyl ester hydrochloride (0.2 mmol), and 1 ml of dichloromethanewas added 122.5 μl (0.7 mmol) of ethyl-diisopropyl-amine with stirring.Stirring was continued at ambient temperature for 40 h. The product wasisolated from the reaction mixture by flash column chromatography onsilica gel using hexanes:ethyl acetate (9:1) as the eluent to give acolorless oil. 27 mg of the title compound was obtained; MS-(+)-ion:M+1=422.8 amu.

f.(R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-hydroxy-propionicacid

A mixture of 27 mg (0.06 mmol) of(R)-3-tert-Butoxy-2-[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid tert-butyl ester and 2 ml of trifluoroacetic acid was stirred for 2h at ambient temperature. Then the excess trifluoroacetic acid wasevaporated in vacuo, the residue dissolved in 2 ml of absolute ethanoland the solution was concentrated in vacuo to give a tan solid. 27 mg ofthe title compound was obtained; MS-(+)-ion: M+1=310.9 amu.

Example A-3(S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-hydroxy-propionicacid

Prepared in analogy to Example A-2 e) and f) from1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid from Example A-2 d)and (S)-2-amino-3-tert-butoxy-propionic acid tert-butyl esterhydrochloride; MS-(+)-ion: M+1=310.9 amu.

Example A-4(R)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-hydroxy-propionicacid

Prepared in analogy to Example A-2 e) and f) from1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylic acid (can beobtained according to U.S. Pat. No. 6,093,730, October 1998, Weidmann etal.) and (R)-2-amino-3-tert-butoxy-propionic acid tert-butyl esterhydrochloride; MS-(+)-ion: M+1=369.0 amu.

Example A-5(S)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-hydroxy-propionicacid

Prepared in analogy to Example A-2 e) and f) from1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylic acid (can beobtained according to U.S. Pat. No. 6,093,730, October 1998, Weidmann etal.) and (S)-2-amino-3-tert-butoxy-propionic acid tert-butyl esterhydrochloride; MS-(+)-ion: M+1=369.0 amu.

Example A-6(R)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-hydroxy-propionicacid

Prepared in analogy to Example A-2 e) and f) from1-chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylic acid (can beobtained according to U.S. Pat. No. 6,093,730, October 1998, Weidmann etal.) and (R)-2-amino-3-tert-butoxy-propionic acid tert-butyl esterhydrochloride; MS-(+)-ion: M+1=369.0 amu.

Example A-7

(S)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-hydroxy-propionicacid

Prepared in analogy to Example A-2 e) and f) from1-chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylic acid (can beobtained according to U.S. Pat. No. 6,093,730, October 1998, Weidmann etal.) and (S)-2-amino-3-tert-butoxy-propionic acid tert-butyl esterhydrochloride; MS-(+)-ion: M+1=369.0 amu.

Example A-82-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-2-methyl-propionicacid

Prepared in analogy to Example A-1 a) and b) from1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid from Example A-2 d)and 2-amino-2-methyl-propionic acid methyl ester hydrochloride;MS-(+)-ion: M+1=308.9 amu.

Example A-92-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-2-methyl-propionicacid

Prepared in analogy to Example A-1 a) and b) from1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylic acid (can beobtained according to U.S. Pat. No. 6,093,730, October 1998, Weidmann etal.) and 2-amino-2-methyl-propionic acid methyl ester hydrochloride;MS-(+)-ion: M+1=367.0 amu.

Example A-10(R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-(1H-imidazol-4-yl)-propionicacid; trifluoro-acetic acid salt

Prepared in analogy to Example A-2 e) from1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid from Example A-2 d)and (R)-2-amino-3-(1-trityl-1H-imidazol-4-yl)-propionic acid methylester hydrochloride followed by deprotection in analogy to Example A-1b) and then in analogy to 2 f); MS-(−)-ion: M−1=359.1 amu.

Example A-11(S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-(1H-imidazol-4-yl)-propionicacid; trifluoro-acetic acid salt

Prepared in analogy to Example A-2 e) from1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid from Example A-2 d)and (S)-2-amino-3-(1-trityl-1H-imidazol-4-yl)-propionic acid methylester hydrochloride followed by deprotection in analogy to Example A-1b) and then in analogy to 2 f); MS-(−)-ion: M−1=359.1 amu.

Example A-12(R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyricacid

Prepared in analogy to Example A-1 a) and b); MS-(−)-ion: M−1=321.1 amu.

Example A-13(S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyricacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=323.0 amu.

Example A-14(R)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyricacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=381.1 amu.

Example A-15(S)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyricacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=381.0 amu.

Example A-16(R)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyricacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=381.0 amu.

Example A-17(S)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyricacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=381.0 amu.

Example A-18(S)-2-[(6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyricacid

Prepared in analogy to Example A-1 a) and b); MS-(−)-ion: M−1=429.0 amu.

Example A-19(R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-phenyl-propionicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=371.0 amu.

Example A-20(S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-phenyl-propionicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=371.0 amu.

Example A-21(R)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-phenyl-propionicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=429.0 amu.

Example A-22(S)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-phenyl-propionicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=429.0 amu.

Example A-23(R)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-phenyl-propionicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=429.0 amu.

Example A-24(S)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-phenyl-propionicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=429.0 amu.

Example A-25(R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-(4-hydroxy-phenyl)-propionicacid

Prepared in analogy to Example A-2 e) and f); MS-(−)-ion: M−1=385.0 amu.

Example A-26(S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-(4-hydroxy-phenyl)-propionicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=387.1 amu.

Example A-27(R)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-(4-hydroxy-phenyl)-propionicacid

Prepared in analogy to Example A-2 e) and f); MS-(−)-ion: M−1=443.0 amu.

Example A-28(S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-3-(4-hydroxy-phenyl)-propionicacid

Prepared in analogy to Example A-2 e) and f); MS-(−)-ion: M−1=443.0 amu.

Example A-29(R)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-(4-hydroxy-phenyl)-propionicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=445.1 amu.

Example A-30(S)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-3-(4-hydroxy-phenyl)-propionicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=445.1 amu.

Example A-31(R)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-pentanoicacid

Prepared in analogy to Example A-1 a) and b); MS-(+)-ion: M+1=381.0 amu.

Example A-32(S)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-pentanoicacid

Prepared in analogy to Example A-1 a) and b); MS-(−)-ion: M−1=379.0 amu.

Example A-33(R)-1-(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-pyrrolidine-2-carboxylicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=321.0 amu.

Example A-34(S)-1-(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-pyrrolidine-2-carboxylicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=321.0 amu.

Example A-35(R)-1-(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-pyrrolidine-2-carboxylicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=379.1 amu.

Example A-36(S)-1-(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-pyrrolidine-2-carboxylicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=379.1 amu.

Example A-37(R)-6-Amino-2-[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-hexanoicacid; trifluoroacetic acid salt

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=352.2 amu.

Example A-38(S)-6-Amino-2-[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-hexanoicacid; trifluoroacetic acid salt

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=352.1 amu.

Example A-39(R)-6-Amino-2-[(1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-hexanoicacid; trifluoroacetic acid salt

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=410.1 amu.

Example A-40(S)-6-Amino-2-[(1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-hexanoicacid; trifluoroacetic acid salt

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=410.1 amu.

Example A-41(R)-6-Amino-2-[(1-chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-hexanoicacid; trifluoroacetic acid salt

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=410.1 amu.

Example A-42(S)-6-Amino-2-[(1-chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-hexanoicacid; trifluoroacetic acid salt

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=410.1 amu.

Example A-43(R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-succinic acid

Prepared in analogy to Example A-1 a) and b); MS-(+)-ion: M+1=338.9 amu.

Example A-44(S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-succinic acid

Prepared in analogy to Example A-2 e) and f); MS-(−)-ion: M−1=337.0 amu.

Example A-45(R)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-succinicacid

Prepared in analogy to Example A-1 a) and b); MS-(+)-ion: M+1=397.0 amu.

Example A-46(S)-2-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-succinicacid

Prepared in analogy to Example A-2 e) and f); MS-(+)-ion: M+1=397.1 amu.

Example A-47(R)-2-[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-succinicacid

Prepared in analogy to Example A-1 a) and b); MS-(+)-ion: M+1=397.0 amu.

Example A-481-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-cyclopropanecarboxylicacid

Prepared in analogy to Example A-1 a) and b); MS-(−)-ion: M−1=305.0 amu.

Example A-491-[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-cyclopropanecarboxylicacid

Prepared in analogy to Example A-1 a) and b); MS-(+)-ion: M+1=365.0 amu.

Example A-50Dideutero-[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

A mixture of 70 mg (0.25 mmol) of1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester fromExample A-2c), 193 mg (2.5 mmol) of glycine-2,2-d₂, and 5 ml of a 0.5Nsodium methoxide solution in methanol was refluxed with stirring for 15h. Then the solvent was evaporated in vacuo, the residue dissolved in 8ml of water, and the solution was washed with 2×20 ml of ethyl acetate.The pH of the solution was adjusted to 3 by addition of aqueous 1Nhydrochloric acid and the mixture was extracted with 3×20 ml of ethylacetate. The combined extracts were dried over magnesium sulfate andconcentrated in vacuo to give a white solid. 61 mg of the title compoundwere obtained; MS-(−)-ion: M−1=280.9 amu.

Example A-51(R)-2-[(6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid a.(R)-2-[(6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl-amino]-propionicacid methyl ester

6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid, 0.33 g,was coupled with D-Alanine methyl ester hydrochloride, 0.150 g,analogously to Example A-1a). 0.205 g of off-white, solid product wereobtained, MS-(+)-ion, M+1=415.0 amu.

b.(R)-2-[(6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid

0.164 g of white solid, prepared analogously to Example A-1 b):MS-(=)-ion, M+1=401.1 amu.

Example A-52(S)-2-[(7-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid a.(S)-2-[(7-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid methyl ester

7-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid, 0.33 g,was coupled with L-Alanine methyl ester hydrochloride, 0.150 g,analogously to Example A-1 a). 0.264 G of white solid were obtained:MS-(+)-ion, M+1=415. amu.

b.(S)-2-[(7-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid

0.216 g of white solid, prepared analogously to Example A-1 b):MS-(+)-ion, M+1=401.9 amu.

Example A-53(R)-2-[(7-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid a.(R)-2-[(7-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid methyl ester

7-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid, 0.33 g,was coupled with D-Alanine methyl ester analogously to Example A-1 a).0.246 g of off-white solid were obtained: MS-(+)-ion, M+1=415.0 amu.

b.(R)-2-[(7-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid

0.211 g of an off-white solid, prepared analogously to Example A-1 b):MS-(+)-ion, M+1=401.0 amu.

Example A-54(S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid a)(S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid methyl ester

1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid, 0.55 g, 1.5 ml oftriethylamine, 0.55 g of DECI, and 0.56 g of (L)-Alanine methyl esterhydrochloride were stirred in 15 ml of methylene chloride at roomtemperature for 72 h. The reaction mixture was partitioned between ethylacetate and water, the organic layer was separated and successivelywashed with 1M aqueous HCl, satd. aqueous NaHCO₃, and satd. aqueousNaCl. The organic layer was dried with sodium sulfate, filtered, andconcentrated under vacuum to afford 0.133 g of off-white solid product:MS-(+)-ion, M+1=308.9 Daltons.

b) (S)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid

0.116 g of (S) methyl ester, described in Example A-54 a), weresaponified/acidified analogously to Example A-1 b) to give 0.087 g of awhite solid product: MS-(+)-ion, M+1=294.9 amu.

Example A-55(R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid a.(R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid methyl ester

1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid, 0.55 g, was coupledwith 0.40 g of D-Alanine methyl ester analogously to Example A-54 a) and0.200 g of off-white, solid product were obtained: MS-(+)-icon,M+1=308.8 amu.

b. (R)-2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid

0.127 g of white solid, prepared analogously to Example A-1 b):MS-(+)-ion, M+1=294.9 amu.

Example A-56(S)-2-[(6-Isopropoxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amimo]-4-propionicacid

0.030 g of 6-isopropoxy-1-chloro-4-hydroxy-isoquinoline-3-carboxylicacid and 0.046 g of HATU were allowed to react with 0.017 g of L-Alaninemethyl ester under analogous conditions to Example A-1 a). Treatment ofthe crude product ester with 0.014 g of NaOH in 0.1 ml of 1:1methanol-water at room temperature for 2 days, followed by acidificationto pH=˜2 with 1M hydrochloric acid, gave a solid product. The productwas collected by filtration, washed with water, and dried to give 0.023g of an off-white solid: MS-(−)-ion, M−1=353.0 amu.

Example A-57(R)-2-[6-Isopropoxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid

Analogously to Example A-56, 0.030 g of6-isopropoxy-1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid wascoupled with D-Alanine methyl ester hydrochloride and the product washydrolyzed to give 0.022 g of an off-white solid: MS-(−)-ion, M−1=353.0amu.

Example A-58(S)-2-[(7-Isopropoxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino-propionicacid

Analogously to Example A-56, 0.040 g of7-isopropoxy-1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid wereallowed to react with 0.020 g of L-Alanine methyl ester hydrochloride togive, after hydrolysis of the intermediate ester, 0.047 g of a whitesolid: MS-(−)-ion, M−1=353.1 amu.

Example A-59(R)-2-[(7-Isopropoxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]propionicacid

Analogously to Example A-56, 0.040 g of7-isopropoxy-1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid wereallowed to react with D-Alanine methyl ester hydrochloride. Theintermediate ester product was hydrolyzed as in Example A-56 to give0.042 g of a white solid: MS-(−)-ion, M−1=353.0 amu.

Example A-602-(S)-{[7-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionicacid a) 4-(4-Chloro-phenoxy)-phthalonitrile

A mixture of 4-nitrophthalonitrile (5.0 g), 4-chlorophenol (3.13 ml) andpotassium carbonate (7.99 g) in acetone (87 ml) was refluxed for 3 h.After filtration and concentration, the residue was dissolved in ethylacetate (100 ml). The solution was washed with NaOH (1N, 50 ml×3) andbrine. The organic layer was dried, filtered, concentrated and dilutedwith dichloromethane. Filtration and rinse through a pad of silica gelgave 5.7 g of the title compound. ¹H NMR (200 MHz, DMSO) δ 8.09 (d, J=9Hz, 1H), 7.83 (d, J=2.6, 1H), 7.53 (d, J=8.6 Hz, 2H), 7.42 (dd, J=2.8,8.6 Hz, 1H), 7.24 (d, J=8.6, 2H).

b) 4-(4-Chloro-phenoxy)-phthalic acid

A mixture of 1.31 g of 4-(4-Chloro-phenoxy)-phthalonitrile, 45%potassium hydroxide (3.5 ml), and methanol (3.5 ml) was refluxed 18 h.6N HCl was added to adjust pH to 4. The precipitate was filtered, washedwith water, and dried to give 1.45 g of the title compound. MS-(−) -ion:M−1=291.0.

c) [5-(4-Chloro-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid butyl ester

A mixture of 500 mg 4-(4-Chloro-phenoxy)-phthalic acid and glycinen-butyl ester (286 mg) was heated at 250° C. for 5 min. The reactionmixture was purified by chromatography with dichloromethane as eluent togive 436 mg the title compound. ¹H NMR (200 MHz, DMSO) δ 7.48 (d, J=8.6Hz, 1H), 7.59 (d, J=9.0 Hz, 2H), 7.46 (m, 2H), 7.29 (d, J=9.0 Hz, 2H),4.46 (s, 2H), 4.16 (t, J=6.2 Hz, 2H), 1.61 (m, 2H), 1.38 (m, 2H), 0.92(t, J=7.0 Hz, 3H).

d) 6- and 7-(4-Chloro-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example A-2 b). Mixture of two isomers.MS-(−)-ion: M−1=386.1.

e) 1-Chloro-6- and7-(4-chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester

Prepared in analogy to Example A-2 c). Mixture of two isomers.MS-(−)-ion: M−1=404.2.

f) 6- and 7-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

A mixture of 1-Chloro-6- and7-(4-chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (280 mg), 0.27 ml of 57 wt % HI, glacial acetic acid (3 ml), andred phosphorous (43 mg) was refluxed for 25 min. Then the mixture wasdiluted with water, basified by solid NaHCO₃ to pH 8, extracted withethyl acetate (2×). The ethyl acetate layer was washed with sodiummetabisulfite solution, saturated sodium bicarbonate, dried andconcentrated. Purification by chromatography with hexanes/ethyl acetategave 7-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (103 mg, Compound of Example A-60 a): MS-(−)-ion: M−1=370.3 and6-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (71 mg, Compound of Example 60b): MS-(−)-ion: M−1=370.3.

g)2-(S)-{[7-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionicacid

Prepared in analogy to Example A-50 by reacting7-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (compound of example A-60 a) with L-alanine in a microwave reactorfor 20 min at 130 C. MS-(−)-ion: M−1=385.1.

Example A-612-(S)-{[6-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionicacid

Prepared in analogy to Example A-50 by reacting6-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (compound of Example A-60 b) with L-alanine in a microwave reactorfor 25 min at 130° C. MS-(−)-ion: M−1=385.1

Example A-622-{[7-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]amino}-propionicacid a) 5-(3,4-Difluoro-phenoxy)-isoindole-1,3-dione

3,4-Difluorophenol (650 mg) was azeotroped with benzene and dissolved insodium methoxide solution in methanol (0.5 M, 10 ml). The methanol wasthen removed under reduced pressure under nitrogen. Then an anhydrousDMF (10 ml) solution of 4-nitrophthalimide (769 mg) was added to theprevious mixture. The resulting mixture was refluxed under nitrogen for23 h. The reaction was cooled down and added 80 ml water. The resultingprecipitate was filtered, washed with water (4×) and dried to give thetitle compound 685 mg. MS-(−)-ion: M−1=274.3.

b) [5-(3,4-Difluoro-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

To a pressure tube was added5-(3,4-difluoro-phenoxy)-isoindole-1,3-dione (680 mg), potassiumcarbonate (1 g), 3-pentanone (20 ml), and methyl bromoacetate (295 μL).The resulting mixture was heated to 105° C. for 17 h. The reaction wasdiluted with 20 ml water and extracted with ethyl acetate (2×). Theorganic layer was dried and concentrated. The mixture was purifiedthrough silica gel chromatography with 4:1 hexanes/ethyl acetate and 3:1hexanes/ethyl acetate to give 657 mg title compound. ¹H NMR (200 MHz,DMSO) δ 7.95 (d, J=9.0 Hz, 1H), 7.64-7.41 (m, 4H), 7.15-7.08 (m, 1H),4.44 (s, 2H), 3.70 (s, 3H).

c) 6- and7-(3,4-Difluoro-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to example A-2 b). Mixture of two isomers.MS-(−)-ion: M−1=388.1.

d) 1-Chloro-6- and7-(3,4-difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester

Prepared in analogy to Example A-2 c). Mixture of two isomers wasdirectly carried on to next step.

e) 6- and 7-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

To a solution of 1-Chloro-6- and7-(3,4-difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (220 mg) in ethyl acetate (4 ml) was added 10% Pd/C (50% wet, 88mg) and then ammonium formate (340 mg). Resulting mixture was heated toreflux for 0.5 h. After cooling, the reaction mixture was diluted withethyl acetate and filtered through a pad of Celite. Filtrate wasconcentrated and separated by chromatography to give 131 mg7-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (Compound of Example A-62 a) and 55 mg6-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (Compound of Example A-62 b).

f)2-{[7-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionicacid

Prepared in analogy to Example A-50 by reacting7-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (compound of Example A-62 a) with L-alanine in a pressure tube for3 days at 85° C. MS-(+)-ion: M−1=389.2.

Example A-632-(S)-[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-propionicacid a) 4-Phenylsulfanyl-phthalic acid

A mixture of 5.06 g of 4-phenylsulfanyl-phthalonitrile (21.4 mmol), 10ml of 50% aqueous KOH, and 10 ml of methanol was refluxed with stirringfor 3.5 days. Then the mixture was diluted with 100 ml of water andacidified with concentrated hydrochloric acid. The precipitated productwas filtered by suction, washed thoroughly with water, and dried invacuo at 60° C. 5.75 g of the title compound were obtained; MS-(−)-ion:M−1=273.0.

b) (1,3-Dioxo-5-phenylsulfanyl-1,3-dihydro-isoindol-2-yl)-acetic acid

5.62 g of 4-phenylsulfanyl-phthalic acid (20.5 mmol) and 1.55 g ofglycine (20.5 mmol) were ground thoroughly together in a mortar. Thenthe mixture was heated to 210° C. to 220° C. in an oil bath. The moltenmass was stirred with a spatula at this temperature for 15 min before itwas allowed to cool to ambient temperature in vacuo. 6.30 g of the titlecompound were obtained; MS-(−)-ion: M−1=311.8; ¹H NMR (DMSO-d₆): δ=7.82(d, 1H), 7.46 to 7.62 (m, 7 H), 4.26 (s, 2H).

c) (1,3-Dioxo-5-phenylsulfanyl-1,3-dihydro-isoindol-2-yl)-acetic acidmethyl ester

A mixture of 20 ml of methanol, 6.27 g of(1,3-dioxo-5-phenylsulfanyl-1,3-dihydro-isoindol-2-yl)-acetic acid (20mmol) and 0.3 ml of concentrated sulfuric acid was refluxed withstirring for 18 h. Then 100 ml of concentrated aqueous sodiumbicarbonate solution were added and the mixture was extracted with 100ml of ethyl acetate. The organic phase was dried over MgSO₄ andevaporated in vacuo. 6.30 g of the title compound were obtained;MS-(+)-ion: M+1=328.0; ¹H NMR (CDCl₃): δ=7.69 (d, 1H), 7.41 to 7.55 (m,7H), 4.40 (s, 2H), 3.75 (s, 3H).

d) 1,4-Dihydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butylester (A) and 1,4-Dihydroxy-6-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester (B)

0.92 g of sodium (40 mmol) were dissolved in 100 ml of n-butanol withstirring. Then the temperature was raised to 95° C. to 100° C., a hotsolution of 6.5 g of(1,3-dioxo-5-phenylsulfanyl-1,3-dihydro-isoindol-2-yl)-acetic acidmethyl ester (19.85 mmol) in 20 ml of n-butanol was added and stirringwas continued at 95° C. to 100° C. for 1 h. Subsequently, the solventwas evaporated in vacuo, 25 ml of aqueous 2N HCl and 100 ml of ethylacetate were added and the mixture was stirred vigorously for 1 h beforeit was filtered by suction. The filter cake was washed thoroughly withwater, and dried in vacuo at 60° C. to give 4.43 g of a yellow solid.4.4 g of this mixture of A and B were separated by flash columnchromatography on silica gel eluting with dichloromethane: ethyl acetate(98:2). Evaporation of the first fraction yielded 1.99 g of A; ¹H NMR(CDCl₃): δ=10.48 (bs, 1H), 8.39 (bs, 1H), 8.24 (d, 1H), 8.01 (d, 1H),7.35 to 7.55 (m, 6H), 4.39 (t, 2H), 1.77 (m, 2H), 1.46 (m, 2H), 0.99 (t,3H). Evaporation of the second fraction yielded 2.26 g of B; ¹H NMR(CDCl₃): δ=10.38 (bs, 1H), 8.32 (bs, 1H), 8.24 (d, 1H), 7.86 (d, 1H),7.37 to 7.56 (m, 6H), 4.39 (t, 2H), 1.77 (m, 2H), 1.46 (m, 2H), 0.99 (t,3H).

e) 1-Bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acidbutyl ester

To a solution of 4.59 g of phosphorous oxybromide (16 mmol) in 25 ml ofanhydrous acetonitrile were added 1.11 g of1,4-dihydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butylester (3 mmol) and the mixture was refluxed gently with stirring for 1h. Then 5.04 g of sodium bicarbonate (60 mmol) were added, followed bythe dropwise addition of 8 ml of water. After stirring at ambienttemperature for 90 min the mixture was concentrated in vacuo to aboutone third of its volume, 40 ml of water were added and the mixture wasextracted with 30 ml of ethyl acetate. The mixture was filtered bysuction. The organic phase was separated, dried over MgSO₄, and filteredthrough a pad of silica gel. Evaporation in vacuo gave 0.885 g of thetitle compound; ¹H NMR (CDCl₃): δ=11.84 (s, 1H), 8.21 (d, 1H), 7.91 (d,1H), 7.40 to 7.55 (m, 6H), 4.46 (t, 2H), 1.84 (m, 2H), 1.48 (m, 2H),0.98 (t, 3H).

f) 4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butyl ester

A mixture of 432 mg of1-bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butylester (1 mmol), 63 mg of red phosphorous (2 mmol), 0.4 ml of aqueous 57wt % HI (3 mmol), and 1 ml of glacial acetic acid was refluxed withstirring for 30 min. Then the reaction mixture was diluted with 25 ml ofethyl acetate, filtered by suction through a pad of celite, washed witha solution of 0.2 g of NaHSO₃ in 5 ml of water, and washed two timeswith 5 ml of concentrated aqueous sodium bicarbonate solution. Theorganic phase was dried over MgSO₄ and evaporated in vacuo. The residuewas purified by flash column chromatography on silica gel eluting withhexanes:ethyl acetate (85:15). 123 mg of the title compound wereobtained; ¹H NMR (CDCl₃): δ=11.85 (s, 1H), 8.60 (s, 1H), 8.23 (d, 1H),7.38 to 7.63 (m, 7H), 4.49 (t, 2H), 1.87 (m, 2H), 1.47 (m, 2H), 0.98 (t,3H).

g)2-(S)-[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-propionicacid

A mixture of 4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acidbutyl ester (0.20 g) and L-alanine (0.75 g) in 0.5 M NaOMe/MeOH (11.3ml) was heated to reflux for 36 h. After cooing, reaction mixture wasconcentrated. The residue was suspended in water (50 ml) and extractedwith ethyl acetate (50 ml) which was discarded. The aqueous layer wasacidified by 2 N HCl aqueous solution. Extracted with ethyl acetate(2×50 ml). Combined organic layers were dried over magnesium sulfate,filtered, and concentrated to give the title compound (0.15 g).MS-(−)-ion: M−1=367.1.

Example A-642-(R)-[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-propionicacid

Prepared in analogy to Example A-63 g) by reacting4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butyl esterwith D-alanine. MS-(−)-ion: M−1=367.1.

Example A-652-(R)-[(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid a) 4-Phenoxy-phthalic acid

Synthesized from 4-phenoxy-phthalonitrile in analogy to Example A-63 a);MS-(−)-ion: M−1=256.9; ¹H NMR (DMSO-d₆): δ=7.93 (d, 1H), 7.07 to 7.52(m, 7H).

b) (1,3-Dioxo-5-phenoxy-1,3-dihydro-isoindol-2-yl)-acetic acid

Synthesized from 4-phenoxy-phthalic acid in analogy to Example A-63 b).MS-(+)-ion: M+1=297.9; ¹H NMR (DMSO-d₆): δ=7.87 (d, 1H), 7.17 to 7.52(m, 7H), 4.26 (s, 2H).

c) (1,3-Dioxo-5-phenoxy-1,3-dihydro-isoindol-2-yl)-acetic acid methylester

Synthesized from (1,3-dioxo-5-phenoxy-1,3-dihydro-isoindol-2-yl)-aceticacid in analogy to Example A-63 c); ¹H NMR (CDCl₃): δ=7.83 (d, 1H), 7.05to 7.46 (m, 7H), 4.41 (s, 2H), 3.76 (s, 3H).

d) 1,4-Dihydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butyl ester(A) and 1,4-Dihydroxy-6-phenoxy-isoquinoline-3-carboxylic acid butylester (B)

Synthesized from (1,3-dioxo-5-phenoxy-1,3-dihydro-isoindol-2-yl)-aceticacid methyl ester in analogy to Example A-63 d); Compound A: ¹H NMR(CDCl₃): δ=10.58 (bs, 1H), 8.37 (bs, 1H), 8.14 (d, 1H), 7.87 (d, 1H),7.05 to 7.49 (m, 6H), 4.39 (t, 2H), 1.77 (m, 2H), 1.46 (m, 2H), 0.99 (t,3H); Compound B: ¹H NMR (CDCl₃): δ=10.38 (bs, 1H), 8.38 (d, 1H), 8.28(bs, 1H), 7.56 (d, 1H), 7.06 to 7.47 (m, 6H), 4.40 (t, 2H), 1.77 (m,2H), 1.46 (m, 2H), 0.99 (t, 3H).

e) 1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butylester

Synthesized from 1,4-dihydroxy-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester in analogy to Example A-63 e); ¹H NMR (CDCl₃): δ=11.89 (s,1H), 8.35 (d, 1H), 7.63 (d, 1 H), 7.08 to 7.52 (m, 6H), 4.47 (t, 2H),1.84 (m, 2H), 1.48 (m, 2H), 0.99 (t, 3H).

f) 4-Hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butyl ester

A mixture of 208 mg of1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butyl ester(0.5 mmol), 49 mg of sodium acetate (0.6 mmol), 50 mg of 10 wt %palladium on charcoal, 10 ml of methanol, and 5 ml of ethyl acetate wasstirred under hydrogen at 1 atm for 15 h. Then the mixture was filteredby suction through a pad of celite and was concentrated in vacuo. Theresidue was partitioned between 2 ml of half concentrated aqueousbicarbonate solution and 8 ml of ethyl acetate. The organic phase wasdried over MgSO₄. Evaporation in vacuo gave 130 mg of the titlecompound; ¹H NMR (CDCl₃): δ=11.89 (bs, 1H), 8.61 (s, 1H), 8.36 (d, 1H),7.10 to 7.53 (m, 7H), 4.49 (t, 2H), 1.87 (m, 2H), 1.47 (m, 2H), 0.98 (t,3H).

g) 2-(R)-[(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid

Prepared in analogy to Example A-63 g) by reacting4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butyl ester withD-alanine at the reflux condition for 5 days. MS-(−)-ion: M−1=351.1.

Example A-662-(S)-{[4-Hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-propionicacid a) 4-(4-Methoxy-phenoxy)-phthalonitrile

A mixture of 4-nitro-phthalonitrile (4.00 g), 4-methoxy-phenol (3.46 g)and potassium carbonate (6.39 g) in acetone (64 ml) was heated to refluxfor 2 h. Reaction mixture was cooled and filtered. Filtrate wasconcentrated and the residue was dissolved in ethyl acetate (100 ml).The solution was washed with NaOH (1N, 50 ml), water, and then brine.The organic layer was dried over magnesium sulfate, filtered, andconcentrated to give the product (6.14 g). ¹H NMR (200 MHz, CDCl₃) δ6.70 (d, J=7.8 Hz, 1H), 7.21 (m, 2H), 6.96 (m, 4H), 3.84 (s, 3H).

b) 4-(4-Methoxy-phenoxy)-phthalic acid

Prepared in analogy to Example A-63 a). MS-(−)-ion: M−1=286.9.

c) [5-(4-Methoxy-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

Prepared in analogy to examples A-63 b and c). ¹H NMR (200 MHz, CDCl₃) δ7.74 (d, J=8.6 Hz, 1H), 7.25 (m, 2H), 6.98 (m, 4H), 4.40 (s, 2H), 3.83(s, 3H), 3.75 (s, 3H).

d) 6- and 7-(4-Methoxy-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example A-63 d). MS-(+)-ion: M+1=384.10.

e) 6- and7-(4-methoxy-phenoxy)-1-bromo-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example A-63 e). MS-(+)-ion: M+1=448.05, 446.05.

g) 7-(4-Methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (A) and 6-(4-Methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (B)

To a solution of the above compound (2.78 g) in ethyl acetate (50 ml)was added 10 wt % palladium on charcoal (wet) (1.2 g) and then ammoniumformate (5.9 g). Resulting mixture was refluxed for 4 h. After cooling,it was filtered and rinsed with ethyl acetate (100 ml). Filtrate wasconcentrated and the residue was purified by silica gel chromatography(33%-50% ethyl acetate in hexanes) to give7-(4-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (A) (0.74 g) (MS-(+)-ion: M+1=368.16) and6-(4-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (B) (1.11 g) (MS-(+)-ion: M+1=368.17).

h)2-(S)-{[4-Hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-propionicacid

Prepared in analogy to Example A-63 g) from7-(4-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (of Example of A-66 a) and L-alanine. MS-(−)-ion: M-1=381.13.

Example A-672-(S)-[(7-Benzenesulfonyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid a) 7-benzenesulfonyl-4-hydroxy-isoquinoline-3-carboxylic acid butylester

A mixture of 7-benzenesulfanyl-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (Compound 363 f) (165 mg) and m-chloroperoxy benzoic acid(77%) (377 mg) in methylene chloride (5 ml) was stirred at roomtemperature overnight. Reaction mixture was filtered. Filtrate wasdiluted with methylene chloride (20 ml) and washed sequentially withsaturated sodium bicarbonate aqueous solution (2×20 ml), water andbrine. Organic layer was dried over magnesium sulfate, filtered, andconcentrated. The crude product was purified by silica gelchromatography (eluting with 0%-20% ethyl acetate in methylene chloride)to give the title compound 120 mg. MS-(+)-ion: M+1=386.11.

b)2-(S)-[(7-Benzenesulfonyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid

Prepared in analogy to Example A-63 g) from7-benzenesulfonyl-4-hydroxy-isoquinoline-3-carboxylic acid butyl esterand L-alanine. MS-(−)-ion: M−1=399.1.

Example A-68(R)-2-[(4-Hydroxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid a) 1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid

A mixture of 1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester (3.52 g, 8.45 mmol; Example A-65 e) aqueous 2N NaOH (50 ml,100 mmol) and EtOH (50 ml) was refluxed with stirring for 2 h. Then thesolution was concentrated in vacuo to ½ of its volume, diluted withwater (180 ml), and was acidified by addition of aqueous 6N HCl (20 ml).After stirring at ambient temperature for 30 min the resultingsuspension was submitted to vacuum filtration. The filter cake waswashed thoroughly with water and dried in vacuo at 70° C. to give thetitle compound as a white solid (3.05 g); ¹H NMR (DMSO-d6): δ=8.33 (d,1H), 7.20 to 7.61 (m, 7H).

b) 4-Benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carboxylic acidbenzyl ester

To a solution of 1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylicacid (721 mg, 2 mmol) in anhydrous THF (100 ml) was added slowly a 2.5 Msolution of n-BuLi in hexanes (3.2 ml, 8 mmol) at −78° C. with stirring.After stirring for another 5 min MeOCH₂I (357 μl, 4 mmol) was added.Stirring was continued for additional 15 min at −78° C. before water (50ml) and aqueous 6N HCl (1.5 ml) were added. The mixture was allowed towarm up to ambient temperature with stirring, and was then concentratedin vacuo to ca. ⅓ of its volume. Traces of iodine were removed byaddition of sodium-meta-bisulfite before the mixture was extracted withEtOAc (100 ml). The organic phase was dried over MgSO₄ and concentratedin vacuo to give a tan solid (576 mg). A mixture of 570 mg of theaforementioned yellowish solid, benzyl bromide (0.97 ml, 8 mmol), K₂CO₃(2.76 g, 20 mmol) and acetone (40 ml) was refluxed with stirring for 3.5d. Then the mixture was concentrated in vacuo. To the residue was addedwater (15 ml) and the mixture was extracted with EtOAc (60 ml). Theorganic phase was dried over MgSO₄ and concentrated in vacuo to give ayellowish oil. Purification by flash column chromatography on silica gelusing hexanes:EtOAc=75:25 as the eluent gave the title compound asyellow oil (490 mg); MS-(+)-ion: M+1=506.2.

c) 4-Benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carboxylic acid

A mixture of4-Benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carboxylic acidbenzyl ester (480 mg, 0.95 mmol), KOH (325 mg, 5 mmol) and EtOH (10 ml)was stirred at ambient temperature for 48 h before the solvent wasevaporated in vacuo. To the residue was added water (10 ml), the mixturewas acidified by the addition of aqueous 6N HCl and extracted with EtOAc(2×25 ml). The combined organic phases were dried over MgSO₄ andconcentrated in vacuo to give the title compound as a tan solid (355mg); MS-(−)-ion: M−1=414.1.

d)(R)-2-[(4-Benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid tert-butyl ester

To a mixture of4-Benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carboxylic acid (79mg, 0.19 mmol), NEt₃ (56 μl, 0.4 mmol), and CH₂Cl₂ (5 ml) cooled with anice bath was added ClCO₂iBu (26.5 μl, 0.2 mmol) with stirring. Afterstirring for 15 min (R)-alanine tert-butyl ester hydrochloride (36 mg,0.2 mmol) was added and the mixture was allowed to warm up to ambienttemperature overnight with stirring. Subsequently; the mixture wasconcentrated in vacuo. To the residue was added water (10 ml) and a fewdrops of aqueous 6N HCl. The mixture was extracted with EtOAc (2×15 ml).The organic phase was dried over MgSO₄ and concentrated in vacuo.Purification by flash column chromatography on silica gel using EtOAc asthe eluent gave the title compound as a tan oil (88 mg); MS-(+)-ion:M+23=565.2.

e)(R)-[(4-Hydroxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid tert-butyl ester

A mixture of(R)-2-[(4-Benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid tert-butyl ester (81 mg, 0.15 mmol), Pd/C (50 mg, 10 wt % Pd),EtOAc (15 ml) was stirred under a H₂-atmosphere at ambient pressure andtemperature for 18 h. Then the mixture was filtered through a pad ofcelite. Celite and filter cake were washed thoroughly with EtOAc and thecombined organic phases were concentrated in vacuo to give the titlecompound as a tan oil (63 mg); MS-(−)-ion: M−1=451.2.

f)(R)-2-[(4-Hydroxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid

A mixture of(R)-[(4-Hydroxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid tert-butyl ester (59 mg, 0.13 mmol) and trifluoroacetic acid (4 ml)was stirred at ambient temperature for 4 h. Then the mixture wasconcentrated in vacuo and the residue dissolved in EtOH. The solvent wasevaporated in vacuo to give the title compound as a tan solid (52 mg);MS-(+)-ion: M+1=397.1.

Example A-69(S)-2-[(4-Hydroxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid a)(S)-2-[(4-Benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid tert-butyl ester

Synthesized from (S)-alanin tert-butyl ester and4-Benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carboxylic acid(Example A-68 c) in analogy to Example A-68 d); MS-(+)-ion: M+23=565.2.

b)(S)-2-[(4-Hydroxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid tert-butyl ester

Synthesized from(S)-2-[(4-Benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid tert-butyl ester in analogy to Example A-68 e); MS-(−)-ion:M−1=451.2.

c)(S)-2-[(4-Hydroxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid

Synthesized from(S)-2-[(4-Hydroxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid tert-butyl ester in analogy to Example A-68 f); MS-(+)-ion:M+1=397.1.

Example A-70(S)-2-[(4-Mercapto-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid a) 4-Dimethylthiocarbamoyloxy-7-phenoxy-isoquinoline-3-carboxylicacid butyl ester

To a solution of 1.5 g of 4-Hydroxy-7-phenoxy-isoquinoline-3-carboxylicacid butyl ester, Example A-65.f, in 6.3 ml of anhydrous DMF was added578 mg of dimethylthiocarbamoylchloride and 1.5 g of1,4-diazabicyclo[2.2.2]octane. The mixture was stirred overnight at roomtemperature. The mixture was poured into 30 ml of 1 N HCl and extractedthree times with 30 ml portions of ethyl acetate. The organic fractionswere washed with water and brine, dried over anhydrous sodium sulfate,and concentrated to 1.9 g of product; MS (+) m/z 425.27 (M+1)

b) 4-Dimethylcarbamoylsulfanyl-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester

A solution of 1.9 g of4-Dimethylthiocarbamoyloxy-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester in 22 ml of phenyl ether was heated to 190° C. for 2 hours.The solution was concentrated under vacuum to give a crude residue,which was purified by column chromatography on silica gel, eluting theproduct with a gradient of 30-80% ethyl acetate in hexanes to give 1.73g; MS (+) m/z 425.07 (M+1)

c) 4-Mercapto-7-phenoxy-isoquinoline-3-carboxylic acid methyl ester

To a solution of 6.5 ml of 0.5 N sodium methoxide in methanol was added460 mg of4-Dimethylcarbamoylsulfanyl-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester. The resultant solution was heated to 50-60° C. for 8 hours,cooled to room temperature, and diluted with 10 ml water and 7.0 ml 1 NHCl. The resulting yellow precipitate was collected by filtering thesolution through a (medium) porous buchner filter funnel to give 307 mgof product; MS (+) m/z 312.08 (M+1)

d)(S)-2-[(4-Mercapto-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid

To a solution of 6.0 ml of 0.5 M sodium methoxide in methanol was added100 mg of 4-Mercapto-7-phenoxy-isoquinoline-3-carboxylic acid methylester and 286 mg of L-alanine. The mixture was heated to 150° C. for 15minutes using a CEM Discover microwave reactor. The resultant solutionwas acidified to pH 3 with 1 N HCl, diluted with 10 ml water, andextracted with 20 ml of ethyl acetate. The organic fraction was washedwith brine, dried over anhydrous sodium sulfate, and concentrated to 114mg of product; MS (−): m/z 369.07 (M−1).

Example A-71(S)-2-{[1-(4-Chloro-phenylsulfanyl)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionicacid a) 1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

The title compound was prepared from(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid methyl ester inanalogy to examples A-65 c)-e); ¹H NMR (200 MHz, CD₃OD) δ 11.89 (s, 1H),8.41 (m, 1H), 8.25 (m, 1H), 7.84 (m, 2H), 4.49 (t, J=7.0 Hz, 2H), 1.87(m, 2H), 1.47 (m, 2H), 1.00 (t, J=7.2 Hz, 3H).

b) (S)-2-{[1-bromo-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionicacid

400 mg of 1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid butyl esterand 890 mg of (L)-Alanine was suspended in a 20 ml solution of 0.5 M ofsodium methoxide in methanol. The mixture was heated to 160° C. for 12min using a CEM Discover microwave reactor. The resultant solution wasconcentrated to ca. 10 ml, and 0.5 N HCl was added until a pH 3 wasreached. The solution was extracted three times with ethyl acetate, andthe organic fractions dried over sodium sulfate and concentrated to atan solid; MS (−): m/z 337.14 (M−1)

c)(S)-2-{[1-(4-Chloro-phenylsulfanyl)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionicacid

To a solution of 250 mg of(S)-2-{[1-bromo-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionic acidin 0.7 ml of 1-methyl-2-pryrrolidinone was added 433 mg of4-chloro-benzenethiol. The solution was heated at 210° C. for 30 min.using a CEM Discover microwave reactor. The solution was concentratedunder vacuum. The resultant residue was crystallized from methanol toyield 18 mg of a tan solid; MS (−): m/z 401.10 (M−1)

Example A-72(R)-2-{[1-(4-Chloro-phenylsulfanyl)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionicacid

The title compound was prepared from1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester, ExampleA-71 a), and (D)-alanine under conditions analogous to Example A-71.b-c;MS (−): m/z 401.08 (M−1).

Example A-73(S)-2-{[7-(3-Fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionicacid a) 4-(3,4-Difluoro-phenoxy)-phthalonitrile

Prepared in analogy to Example A-60 a). ¹H NMR (200 MHz, DMSO) δ 8.14(d, J=9 Hz, 1H), 7.95 (d, J=2.6, 1H), 7.56 (dd, J=2.6, 8.6 Hz, 1H), 7.19(dt, J=2.4, 9.2 Hz, 1H), 7.04 (m, 2H).

b) 4-(3-Fluoro-5-methoxy-phenoxy)-phthalic acid

Prepared in analogy to Example A-60 b). One of the fluoro group issubstituted by a methoxy group during the hydrolysis. MS-(−)-ionM−1=305.0.

c)[5-(3-Fluoro-5-methoxy-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid butyl ester

Prepared in analogy to Example A-60 c). ¹H NMR (200 MHz, DMSO) δ 7.93(d, J=8.6 Hz, 1H), 7.43 (m, 2H), 6.79-6.63 (m, 3H), 4.41 (s, 2H), 4.10(t, J=6.2, 2H), 1.54 (m, 2H), 1.30 (m, 2H), 0.86 (t, J=7.0, 3H).

d) 6- and7-(3-Fluoro-5-methoxy-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example A-2 b). Mixture of two isomers.MS-(−)-ion M−1=400.1.

e) 1-Chloro-6- and7-(3-fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example A-2 c). Mixture of two isomers.MS-(−)-ion M−1=418.3.

f) 6- and7-(3-Fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to example A-62 e). The mixture of isomers wereseparated to give7-(3-fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (Compound of example A-73 a) and6-(3-fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (Compound of example A-73 b). ¹H NMR (200 MHz, CD₃OD) δ 8.73(s, 1H), 8.15 (d, J=9.0 Hz, 1H), 7.71 (s, 1H), 7.59 (m, 1H), 6.65-6.47(m, 3H), 4.49 (t, J=6.6 Hz, 2H), 3.81 (s, 3H), 1.87 (m, 2H), 1.56 (m,2H), 1.03 (t, J=7.4. 3H).

g)(S)-2-{[7-(3-Fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionicacid

Prepared in analogy to Example A-50 by reacting7-(3-fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (compound of example A-73 a) with L-alanine in a pressuretube for 3 days at 90 C. MS-(−)-ion M−1=399.1.

Example A-742-(S)-[(7-Cyclohexyloxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid a. (5-Hydroxy-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acidethyl ester

Prepared in analogy to example D-100 c) from 4-hydroxy-phthalic acid andglycine ethyl ester HCl salt. ¹H NMR (200 MHz, DMSO-d₆) δ 11.0 (br s,1H), 7.74 (d, J=7.8 Hz, 1H), 7.17 (m, 2H), 4.35 (s, 2H), 4.13 (q, J=7.0Hz, 2H), 1.20 (t, J=7.0 Hz, 3H).

b. (5-Cyclohexyloxy-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acidethyl ester

To a mixture of (5-hydroxy-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-aceticacid ethyl ester (8.0 g) in anhydrous tetrahydrofuran (160 ml) was addedcyclohexanol (3.2 g), diethylazadicarboxylate (6.9 g) and then triphenylphosphine (12.6 g). Resulting mixture was stirred at room temperatureovernight and concentrated. Residue was partitioned between water andethyl acetate. Aqueous layer was extracted with ethyl acetate. Combinedorganic layers were washed with brine, dried over magnesium sulfate andfiltered. Filtrate was concentrated and purified by silica gelchromatography (eluting with 5% ethyl acetate in methylene chloride) togive the title compound (6.2 g). ¹H NMR (200 MHz, CDCl₃) δ 7.73 (dd,J=8.2, 0.8 Hz, 1H), 7.30 (br s, 1H), 7.12 (m, 1H), 4.38 (m, 3H), 4.21(q, J=7.1 Hz, 2H), 2.02 (m, 2H), 1.82-1.25 (m, 13H).

c. 6- and 7-Cyclohexyloxy-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example A-63 d) to give7-cyclohexyloxy-1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound A-74 c1) (MS-(+)-ion M+1=360.16) and6-cyclohexyloxy-1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound A-74 c2) (MS-(+)-ion M+1=360.18).

d. 1-Bromo-7-cyclohexyloxy-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

A mixture of 7-cyclohexyloxy-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound A-74 c1) (1.3 g) and phosphorus oxybromide(1.35 g) in anhydrous toluene (25 ml) was heated in a microwave reactor(sealed tube) at 130° C. for 15 min. After cooling, reaction mixture wasconcentrated. The residue was treated with saturated sodium bicarbonateaqueous solution (100 ml) and stirred at room temperature for 20 min.Extracted with ethyl acetate. Organic layer was washed with water,brine, dried over magnesium sulfate, filtered, and concentrated to givethe title compound (1.2 g). MS-(+)-ion M+1=422.12, 424.12.

e. 7-Cyclohexyloxy-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

To a mixture of1-bromo-7-cyclohexyloxy-4-hydroxy-isoquinoline-3-carboxylic acid butylester (936 mg) in ethyl acetate (25 ml) was added 10% Pd/C (50% wet)(430 mg) and then ammonium formate (1.4 g). Resulting mixture wasrefluxed for 4 h. After cooling, reaction mixture was filtered andconcentrated. The residue was purified by silica gel chromatography(3%-10% ethyl acetate in methylene chloride) to give the title compound(550 mg). MS-(+)-ion M+1=344.22.

f.2-(S)-[(7-Cyclohexyloxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid

A mixture of 7-cyclohexyloxyl-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (80 mg) and L-alanine (207 mg) in 0.5 M sodium methoxide inmethanol (3.7 ml) was heated in a microwave reactor (sealed tube) at120° C. for 40 min. Reaction mixture was concentrated, dissolved inwater (30 ml), and acidified by 2 N HCl to pH=4. It was extracted withethyl acetate. Organic layer was washed with water, brine, dried overmagnesium sulfate, and filtered. Filtrate was concentrated and purifiedby silica gel chromatography to give the title compound (52 mg).MS-(+)-ion M+1=359.18.

Example A-752-(S)-{[7-(4-Fluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-propionicacid a. 5-(4-Fluoro-phenoxy)-isoindole-1,3-dione

A mixture of 5-Nitro-isoindole-1,3-dione (177 g, 0.904 mol),4-fluoro-phenol (128 g, 1.13 mol), K₂CO₃ (419 g, 3 mol) and DMF (2 l)was refluxed with stirring for 3 h before the mixture was poured intowater (12 l) with stirring. The precipitate formed was isolated byvacuum filtration, washed with water (8 l) and dried in vacuo at 70° C.to give the title compound as a tan powder (43.2 g); ¹H NMR (CDCl₃)δ=7.79 (d, 1H), 7.57 (br s, 1H), 7.01 to 7.29 (m, 6H).

b. [5-(4-Fluoro-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

A mixture of 5-(4-fluoro-phenoxy)-isoindole-1,3-dione (42.9 g, 167mmol), Bromo-acetic acid methyl ester (21.1 ml, 223 mmol), K₂CO₃ (62.3g, 446 mmol) and Et₂CO (700 ml) was refluxed with stirring for 16 hbefore the mixture was concentrated in vacuo. To the residue was addedwater (150 ml) and the resulting slurry was extracted with EtOAc (1×750ml, 1×250 ml). The combined organic phases were dried over MgSO₄ andconcentrated in vacuo to give the title compound as a tan solid (49.7g); ¹H NMR (CDCl₃) δ=7.80 (d, 1H), 7.01 to 7.30 (m, 6H), 4.41 (s, 2H),3.76 (s, 3H).

c. 7-(4-Fluoro-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester

Sodium (7.2 g, 310 mmol) was dissolved in n-butanol (300 ml) withstirring at 70° C. Afterwards, the temperature was raised to 95-100° C.and a solution of[5-(4-Fluoro-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-acetic acidmethyl ester (49.4 g, 150 mmol) in hot n-butanol (300 ml) was added withvigorous stirring. The mixture was stirred for another 90 min at 95-100°C. and was then allowed to cool to 60° C. with stirring before 2 N HCl(160 ml) was added. The mixture was stirred vigorously for 30 min andwas then allowed to cool to ambient temperature. Subsequently, themixture was submitted to vacuum filtration. The filter cake was washedthoroughly with water and dried in vacuo at 70° C. to give a pale yellowsolid. Purification by flash column chromatography on silica gel usingCH₂Cl₂ EtOAc=98.2 as the eluent gave the title compound (14.4 g, firstfraction); ¹H NMR (CDCl₃) δ=8.40 (br s, 1H), 8.14 (d, 1H), 7.80 (d, 1H),7.42 to 7.48 (m, 1H), 7.04 to 7.14 (m, 4H), 4.39 (t, 2H), 1.70 to 1.85(m, 2H), 1.37 to 1.55 (m, 2H), 0.99 (t, 3H).

d. 1-Bromo-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

A mixture of7-(4-Fluoro-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acid butylester (14.33 g, 38.6 mmol), POBr₃ (44.7 g, 154.4 mmol) and anhydrousmethyl cyanide (290 ml) was refluxed gently with stirring for 75 minbefore NaHCO₃ (100.8 g, 1.2 mol) was added in small portions withstirring. Subsequently, water (200 ml) was added slowly with stirringand the mixture was stirred vigorously for 1 h at ambient temperaturebefore it was concentrated in vacuo to ca. ½ of its volume. Then water(200 ml) was added and the mixture was extracted with EtOAc (1×400 ml,1×200 ml). The combined organic phases were dried over MgSO₄ andevaporated in vacuo to give a tan solid. The tan solid was dissolved inCH₂Cl₂ and purified by filtration through a plug of silica gel. In vacuoconcentration of the resulting CH₂Cl₂ solution yielded the titlecompound (11.4 g); ¹H NMR (CDCl₃) δ=11.89 (s, 1H), 8.36 (d, 1H), 7.57(d, 1H), 7.44 to 7.50 (m, 1H), 7.08 to 7.16 (m, 4H), 4.47 (t, 2H), 1.78to 1.93 (m, 2H), 1.38 to 1.58 (m, 2H), 0.99 (t, 3H).

e. 7-(4-Fluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylicacid butyl ester

A mixture of1-Bromo-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (434 mg, 1 mmol), Pd(PPh₃)₄ (116 mg, 0.1 mmol),trimethylboroxine (140 μl, 1 mmol), K₂CO₃ (414 mg, 3 mmol), and1,4-dioxane (8 ml) was refluxed with stirring for 2 h. Subsequently, themixture was concentrated in vacuo. To the residue was added water (10ml). The mixture was acidified by the addition of aqueous 6N HCl andthen extracted with EtOAc (40 ml). The organic phase was dried overMgSO₄ and evaporated in vacuo. Purification of the residue by flashcolumn chromatography on silica gel using hexanes:EtOAc=94:6 as theeluent gave the title compound as white solid (229 mg); MS-(+)-ionM+1=370.1.

f)2-(S)-{[7-(4-Fluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-propionicacid

A mixture of7-(4-Fluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylic acidbutyl ester (92 mg, 0.25 mmol), (S)-alanine (225 mg, 2.5 mmol) and a 0.5N solution of MeONa in MeOH (5 ml, 2.5 mmol) was heated in a microwaveoven with stirring for 20 min at 140° C. before the mixture wasconcentrated in vacuo. To the residue was added water (10 ml) and themixture was washed with EtOAc (2×25 ml). The so purified aq. solutionwas acidified by the addition of 6 N HCl and extracted with EtOAc (1×25ml). The organic phase was dried over MgSO₄ and concentrated in vacuo togive the title compound as a tan solid (69 mg); MS-(+)-ion M+1=385.1.

Example A-762-(S)-{[7-(4-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionicacid a. 7-(4-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

A mixture of1-Bromo-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (4.34 g, 10 mmol, see example A-75d), sodium acetate (984mg, 12 mmol), Pd/C (2.0 g, 10 wt % Pd, 50 wt % water), EtOAc (400 ml)and MeOH (200 ml) was stirred under an H₂-atmosphere at ambient pressureand temperature for 2.5 h before the mixture was filtered through a plugof celite. The celite was washed with EtOAc (500 ml). The combinedorganic phases were concentrated in vacuo. To the residue was added ahalf concentrated NaHCO₃ solution (50 ml) and the mixture was extractedwith CH₂Cl₂ (1×200 ml). The organic phase was dried over MgSO₄ andconcentrated in vacuo to yield the title compound as a tan oil (3.45 g);MS-(+)-ion M+1=356.1.

b.2-(S)-{[7-(4-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-propionicacid

A mixture of 7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (154 mg, 0.43 mmol), (S)-alanine (225 mg, 2.5 mmol) anda 0.5 N solution of MeONa in MeOH (5 ml, 2.5 mmol) was heated in amicrowave oven with stirring for 20 min at 130° C. before the mixturewas concentrated in vacuo. To the residue was added water (15 ml) andthe mixture was washed with Et₂O (3×30 ml). The purified aq. solutionwas acidified by the addition of 6 N HCl and extracted with EtOAc (1×30ml). The organic phase was dried over MgSO₄ and concentrated in vacuo togive the title compound as a tan solid (79 mg); MS-(−)-ion M−1=369.1.

Example A-772-(S)-[(4-Hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid a. 1-Chloro-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylic acidbutyl ester and 1-Chloro-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylicacid butyl ester (regioisomeric mixture)

To POCl₃ (300 ml) was added a regioisomeric mixture of1,4-dihydroxy-6-phenoxy-isoquinoline-3-carboxylic acid butyl ester and1,4-dihydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butyl ester(40.63 g, 115 mmol, see example A-65d). The mixture was refluxed gentlywith stirring for 30 min before it was concentrated in vacuo. Theresidue was dissolved in EtOAc (800 ml) and water (400 ml) was added. Tothe vigorously stirred mixture was then added NaHCO₃ (ca. 100 g) insmall portions. Subsequently, the mixture was stirred for 1 h at ambienttemperature before it was filtered through a pad of celite. The organicphase was separated, dried over MgSO₄ and evaporated in vacuo to give atan solid. The tan solid was dissolved in CH₂Cl₂ and purified byfiltration through a plug of silica gel. In vacuo concentration of theresulting CH₂Cl₂ solution yielded the title compounds (15.51 g) as a tansolid; MS-(−)-ion M−1=370.2.

b. 4-Hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carboxylic acid butylester

To a mixture of anhydrous 1,4-dioxane (200 ml), Pd(PPh₃)₄ (3.47 g, 3mmol), trimethylboroxine (4.22 ml, 30 mmol), and K₂CO₃ (12.44 g, 90mmol) was added a regioisomeric mixture of1-chloro-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylic acid butyl esterand 1-Chloro-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butylester (11.15 g, 30 mmol). The mixture was refluxed under N₂-protectionwith stirring for 3 h and was then stirred at ambient temperature for 48h. Subsequently, the mixture was concentrated in vacuo. To the residuewas added water (100 ml) and the mixture was extracted with EtOAc (300ml). The organic phase was dried over MgSO₄ and evaporated in vacuo.Purification of the residue by flash column chromatography on silica gelusing hexanes:EtOAc=9:1 as the eluent gave the title compound as ayellowish solid (4.40 g, first fraction); MS-(+)-ion M+1=352.1.

c.2-(S)-[(4-Hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid

A mixture of 4-Hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester (176 mg, 0.5 mmol), (S)-alanine (225 mg, 2.5 mmol) and a 0.5N solution of MeONa in MeOH (5 ml, 2.5 mmol) was heated in a microwaveoven with stirring for 20 min at 120° C. before the mixture wasconcentrated in vacuo. To the residue was added water (15 ml) and themixture was washed with Et₂O (3×30 ml). The so purified aq. solution wasacidified by the addition of 6 N HCl and extracted with EtOAc (1×30 ml).The organic phase was dried over MgSO₄ and concentrated in vacuo to givethe title compound as a tan solid (108 mg); MS-(−)-ion M−1=365.1.

Example A-782-(S)-[(4-Hydroxy-1-methyl-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-propionicacid a) 1-Bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylicacid

1,4-Dihydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butylester (Example A-63 d) Compound A) (29.0 g) and phosphorous oxybromide(67.5 g) in 600 ml anhydrous acetonitrile was stirred at reflux for 4hours. After cooling the reaction mixture was concentrated and saturatedsodium bicarbonate solution and ethyl acetate were added to the residueand stirred overnight. Precipitate that formed between layers wascollected and washed with water to give the title compound (10.2 g).MS-(+)-ion M+1=376.0, 378.1.

b) 1-Bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acidmethyl ester

1-Bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid (10.0g), potassium carbonate (3.7 g) and methyl sulfate (3.4 g) weresuspended in 500 ml acetone and stirred at reflux overnight. Reactionmixture was concentrated and residue partitioned between 1 Nhydrochloric acid and ethyl acetate. Organic layer was dried overmagnesium sulfate and filtered. Filtrate concentrated to give titlecompound (9.6 g). MS-(+)-ion M+1=389.9, 391.9.

c) 4-Hydroxy-1-methyl-7-phenylsulfanyl-isoquinoline-3-carboxylic acidmethyl ester

1-Bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid methylester (0.2 g), tetrakis(triphenylphosphine)palladium (60 mg), methylboroxine (65 mg), and potassium carbonate in 1,4-dioxane (4 ml) wereheated in a microwave reactor (sealed tube) for 10 min at 140° C. Aftercooling reaction mixture was concentrated and partitioned between 1 Nhydrochloric acid and ethyl acetate. Organic layer dried over magnesiumsulfate and filtered. Filtrate concentrated and separated by silica gelchromatography (eluting with 2% ethyl acetate in methylene chloride) togive the title compound (47 mg). MS-(+)-ion M+1=326.1.

d)2-(S)-[(4-Hydroxy-1-methyl-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-propionicacid

Prepared in analogy to Example A-74 f). ¹H NMR (200 MHz, DMSO-d6) δ13.26 (br s, 1H), 9.07 (s, 1H), 8.61 (s, 1H), 8.33 (d, J=8.2 Hz 1H),7.97 (d, J=8.6 Hz, 1H), 7.81 (br s, 2H), 7.52 (br s, 3H), 4.52 (br s,1H), 2.91 (s, 3H), 1.49 (d, J=7.0 Hz, 3H).

Example A-792-(S)-{[4-Hydroxy-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-propionicacid a) 4-(4-Trifluoromethyl-phenoxy)-phthalonitrile

Prepared in analogy to Example A-66 a). ¹H NMR (200 MHz, CDCl₃) δ 7.74(m, 2H), 7.47 (d, J=8.6 Hz, 1H), 7.25 (m, 3H), 6.87 (d, J=8.9 Hz, 1H).

b) 4-(4-Trifluoromethyl-phenoxy)-phthalic acid

Prepared in analogy to Example A-66 b). ¹H NMR (200 MHz, DMSO-d6) δ 8.24(d, J=9.0 Hz, 1H), 7.75 (m, 3H), 7.19 (m, 3H).

c)[1,3-Dioxo-5-(4-trifluoromethyl-phenoxy)-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

Prepared in analogy to Example A-66 c). ¹H NMR (200 MHz, CDCl₃) δ 7.86(d, J=8.5 Hz, 1H), 7.67 (d, J=8.2 Hz, 2H), 7.40-7.13 (m, 4H), 4.43 (s,2H), 3.76 9 s, 3H).

d) 7-(4-trifluoromethyl-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example A-66 d). Two isomers were separated bychromatography to give the title compound. MS-(+)-ion M+1=422.0

e)1-Chloro-4-hydroxy-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example A-2 c). MS-(−)-ion M−1=438.3.

f) 4-Hydroxy-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example A-74 e). MS-(+)-ion M+1=406.1.

g)2-(S)-{[4-Hydroxy-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-propionicacid

Prepared in analogy to Example A-74 f). MS-(+)-ion M+1=421.2.

Example B-1 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid(2-amino-ethyl)-amide; trifluoro-acetic acid salt a.(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid butyl ester

A mixture of 160 ml of butanol, 20.0 g of(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid (94.6 mmol) and 2.0 mlof concentrated sulfuric acid was refluxed with stirring for 24 h. Then5 g of sodium bicarbonate were added in portions, stirring continued atr.t. for 5 min and the solvent evaporated in vacuo. The residue waspartitioned between 100 ml of water and 100 ml of ethyl acetate. Theorganic phase was washed with 100 ml of brine, dried over sodium sulfateand was evaporated in vacuo to give a yellowish oil that latersolidified. 24.02 g of the title compound were obtained; MS-(+)-ion:M+1=261.9.

b. 1,4-Dihydroxy-isoquinoline-3-carboxylic acid butyl ester

4.41 g of sodium (190 mmol) were dissolved in 250 ml of n-butanol withstirring. After the sodium was completely dissolved the solution wasallowed to cool to ambient temperature and a solution of 24.0 g (91.9mmol) of (1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid butyl esterin 150 ml of butanol was added with stirring. The solution was heated to100° C. within 30 min and stirred at this temperature for 1 h. Then themixture was allowed to cool to ambient temperature and was stored atambient temperature for 18 h. Then the pH of the mixture was adjusted to2 to 3 by the addition of aqueous 2N hydrochloric acid with stirring.Stirring was continued for 30 min before the solid component wasfiltered by suction. The filter cake was washed thoroughly with water,and dried in vacuo at 50° C. to give a white solid. 17.75 g of the titlecompound were obtained; MS-(+)-ion: M+1=262.1.

c. 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

A mixture of 17.3 g (66.2 mmol) of1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester and 100 ml ofphosphorous oxychloride was stirred at ambient temperature for 1 h, andthen heated slowly with stirring in the course of 2 h to refluxtemperature. The mixture was refluxed gently with stirring for 30 min.After cooling to room temperature the excess phosphorous oxychloride wasevaporated in vacuo, and the residue was dissolved in 100 ml of ethylacetate. The solution was poured into 300 ml of a saturated aqueoussodium bicarbonate solution with stirring. The precipitate formed wasremoved by vacuum filtration. The organic phase was separated, and theaqueous phase was extracted with 3×100 ml of ethyl acetate. The combinedaqueous phases were dried over sodium sulfate, filtered through a pad ofsilica gel and evaporated in vacuo to give a brown oil that solidifiedlater. 11.37 g of the title compound were obtained; ¹H NMR (CDCl₃):δ=11.91 (s, 1H), 8.41 (m, 1H), 8.29 (m, 1H), 7.83 (m, 2H), 4.49 (t, 2H),1.84 (m, 2H), 1.48 (m, 2H), 0.99 (t, 3H).

d. 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid

A mixture of 9.23 g of 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (33 mmol), 90 ml of 2.5 N aqueous sodium hydroxide solution,water (20 ml) and ethanol (110 ml) was refluxed with stirring for 2 h.Then the pH of the mixture was adjusted to 2 by the addition ofconcentrated aqueous hydrochloric acid. During the addition thetemperature of the mixture was kept at 20° C. by cooling with an icebath. Stirring was then continued for 1 h before the solid component wasseparated by vacuum filtration. The filter cake was washed with waterand dried in vacuo at 85° C. to give a white powder. 6.64 g of the titlecompound were obtained; MS-(+)-ion: M+1=224.1.

e.{2-[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-ethyl}-carbamicacid tert-butyl ester

To a mixture of 45 mg (0.2 mmol) of1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid, 76 mg (0.2 mmol) ofbenzotriazol-1-yl-(bis-dimethylamino-methylene)-oxonium hexafluorophosphate (HBTU), 32 μl (2-amino-ethyl)-carbamic acid tert-butyl ester(0.2 mmol), and 1 ml of dichloromethane was added 96 μl (0.55 mmol) ofethyl-diisopropyl-amine with stirring. Stirring was continued at ambienttemperature for 5 days. The product was isolated from the reactionmixture by flash column chromatography on silica gel using hexanes:ethylacetate (8:2) as the eluent to give a tan gum. 8 mg of the titlecompound were obtained; MS-(−)-ion: M−1=364.0.

f. 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid{2-amino-ethyl}-amide; trifluoro-acetic acid salt

A mixture of 8 mg (0.022 mmol) of{2-[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-ethyl}-carbamicacid tert-butyl ester and 2 ml of trifluoroacetic acid was stirred for 2h at ambient temperature. Then the excess trifluoroacetic acid wasevaporated in vacuo, the residue dissolved in absolute ethanol and thesolution concentrated in vacuo to give a tan solid. 8.5 mg of the titlecompound were obtained; MS-(+)-ion: M+1=266.0.

Example B-2 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid(2-methoxy-ethyl)-amide

To a mixture of 45 mg (0.2 mmol) of1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid (from example A-1 d),76 mg (0.2 mmol) ofbenzotriazol-1-yl-(bis-dimethylamino-methylene)-oxonium hexafluorophosphate (HBTU), 18 μl 2-methoxy-ethylamine (0.2 mmol), and 1 ml ofdichloromethane were added 96 μl (0.55 mmol) of ethyl-diisopropyl-aminewith stirring. Stirring was continued at ambient temperature for 12days. The product was isolated from the reaction mixture by flash columnchromatography on silica gel using hexanes:ethyl acetate (9:1) as theeluent to give a white solid. 8.8 mg of the title compound wereobtained; MS-(+)-ion: M+1=281.0.

Example B-3 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid(2-hydroxy-ethyl)-amide

Synthesized from 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid fromexample A-1 d) and 2-amino-ethanol in analogy to example 2; MS-(−)-ion:M−1=265.2.

Example B-4 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid(2-dimethylamino-ethyl)-amide

A mixture of 28 mg (0.1 mmol) of1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester fromexample A-1 c), 116 μl (1 mmol) of N,N-dimethyl-ethane-1,2-diamine and0.5 ml of absolute ethanol was stirred at ambient temperature for 18 h.Then the solvent was evaporated in vacuo, the residue suspended in 5 mlof water, and mixture was extracted with 2×35 ml of ethyl acetate. Thecombined organic phases were dried over sodium sulfate and evaporated invacuo to give a yellowish solid. 29 mg of the title compound wereobtained; MS-(+)-ion: M+1=294.1.

Example B-5 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid(2-acetylamino-ethyl)-amide

A mixture of 56 mg (0.2 mmol) of1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester fromexample B-1 c), 227 mg (2 mmol) of N-(2-amino-ethyl)-acetamide and 0.8ml of absolute ethanol was stirred at ambient temperature for 3 days.Then the solvent was evaporated in vacuo, the residue suspended in 3 mlof water, and the pH of the mixture was adjusted 2 to 3 by the additionof aqueous 1N HCl. The mixture was extracted with 2×25 ml of ethylacetate. The combined organic phases were dried over sodium sulfate andevaporated in vacuo to give a yellowish solid. 64 mg of the titlecompound were obtained; MS-(+)-ion: M+1=308.1.

Example B-6 1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylicacid (2-hydroxy-ethyl)-amide

Synthesized from1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylic acid butylester (can be obtained according to U.S. Pat. No. 6,093,730, October1998, Weidmann et al.) and 2-amino-ethanol in analogy to example B-5;MS-(+)-ion: M+1=325.1.

Example B-7 1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylicacid (2-methoxy-ethyl)-amide

Synthesized from1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylic acid butylester (can be obtained according to U.S. Pat. No. 6,093,730, October1998, Weidmann et al.) and 2-methoxy-ethylamine in analogy to exampleB-5; MS-(+)-ion: M+1=339.0.

Example B-8 1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylicacid (2-amino-ethyl)-amide; trifluoro-acetic acid salt

Synthesized from1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylic acid butylester (can be obtained according to U.S. Pat. No. 6,093,730, October1998, Weidmann et al.) and (2-amino-ethyl)-carbamic acid tert-butylester in analogy to example B-5, followed by deprotection in analogy toexample B-1 f); MS-(+)-ion: M+1=324.1.

Example B-9 1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylicacid (2-dimethylamino-ethyl)-amide

Synthesized from1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylic acid butylester (can be obtained according to U.S. Pat. No. 6,093,730, October1998, Weidmann et al.) and N,N-dimethyl-ethane-1,2-diamine in analogy toexample B-4, followed by deprotection in analogy to example B-1 f);MS-(+)-ion: M+1=352.1.

Example B-10 1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylicacid (2-amino-ethyl)-amide; trifluoro-acetic acid salt

Synthesized from1-chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylic acid butylester (can be obtained according to U.S. Pat. No. 6,093,730, October1998, Weidmann et al.) and (2-amino-ethyl)-carbamic acid tert-butylester in analogy to example B-5, followed by deprotection in analogy toexample B-1 f); MS-(+)-ion: M+1=324.0.

Example B-11 1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylicacid (2-methoxy-ethyl)-amide

Synthesized from1-chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylic acid butylester (can be obtained according to U.S. Pat. No. 6,093,730, October1998, Weidmann et al.) and 2-methoxy-ethylamine in analogy to exampleB-5; MS-(−)-ion: M−1=337.1.

Example B-12 1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylicacid (2-dimethylamino-ethyl)-amide

Synthesized from1-chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylic acid butylester (can be obtained according to U.S. Pat. No. 6,093,730, October1998, Weidmann et al.) and N,N-dimethyl-ethane-1,2-diamine in analogy toexample B-4, followed by deprotection in analogy to example B-1 f);MS-(+)-ion: M+1=352.1.

Example B-13 1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylicacid (2-hydroxy-ethyl)-amide

Synthesized from1-chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylic acid butylester (can be obtained according to U.S. Pat. No. 6,093,730, October1998, Weidmann et al.) and 2-amino-ethanol in analogy to example B-5;MS-(−)-ion: M−1=323.2.

Example C-1 1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylicacid (2-hydroxy-1-hydroxymethyl-ethyl)-amide

0.035 gm of 1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylicacid butyl ester and 0.088 g of 2-amino-propane-1,3-diol were dissolvedin 1 ml of ethanol and the mixture was refluxed for 24 h. The reactionmixture was concentrated and the residue was dissolved in 10 ml of ethylacetate. The ethyl acetate solution was extracted with 5 ml of aqueous 1M HCl and water, dried (sodium sulfate) and concentrated to give 0.042 gof a white solid: MS-(+)-ion: 355.1.

Example C-2 1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylicacid (2-hydroxy-1-hydroxymethyl-ethyl)-amide

Prepared from 1-chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylicacid butyl ester and 2-amino-propane-1,3-diol analogously to ExampleC-1: MS-(−)-ion: 353.2.

Example C-3 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid(2-hydroxy-1-hydroxymethyl-ethyl)-amide

Prepared from 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butylester and 2-amino-propane-1,3-diol analogously to Example C-1:MS-(−)-ion: 295.2.

Example D-1[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acida) 4-Phenylsulfanyl-phthalic acid

A mixture of 5.06 g of 4-phenylsulfanyl-phthalonitrile (21.4 mmol), 10ml of 50% aqueous KOH, and 10 ml of methanol was refluxed with stirringfor 3.5 days. Then the mixture was diluted with 100 ml of water andacidified with concentrated hydrochloric acid. The precipitated productwas filtered by suction, washed thoroughly with water, and dried invacuo at 60° C. 5.75 g of the title compound were obtained; MS-(−)-ion:M−1=273.0.

b) (1,3-Dioxo-5-phenylsulfanyl-1,3-dihydro-isoindol-2-yl)-acetic acid

5.62 g of 4-phenylsulfanyl-phthalic acid (20.5 mmol) and 1.55 g ofglycine (20.5 mmol) were ground thoroughly together in a mortar. Thenthe mixture was heated to 210° C. to 220° C. in an oil bath. The moltenmass was stirred with a spatula at this temperature for 15 min before itwas allowed to cool to ambient temperature in vacuo. 6.30 g of the titlecompound were obtained; MS-(−)-ion: M−1=311.8.

c) (1,3-Dioxo-5-phenylsulfanyl-1,3-dihydro-isoindol-2-yl)-acetic acidmethyl ester

A mixture of 20 ml of methanol, 6.27 g of(1,3-dioxo-5-phenylsulfanyl-1,3-dihydro-isoindol-2-yl)-acetic acid (20mmol) and 0.3 ml of concentrated sulfuric acid was refluxed withstirring for 18 h. Then 100 ml of concentrated aqueous sodiumbicarbonate solution were added and the mixture was extracted with 100ml of ethyl acetate. The organic phase was dried over MgSO₄ andevaporated in vacuo. 6.54 g of the title compound were obtained;MS-(+)-ion: M+1=328.0.

d) 1,4-Dihydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butylester (A) and 1,4-Dihydroxy-6-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester (B)

0.92 g of sodium (40 mmol) were dissolved in 100 ml of n-butanol withstirring. Then the temperature was raised to 95° C. to 100° C., a hotsolution of 6.5 g of(1,3-dioxo-5-phenylsulfanyl-1,3-dihydro-isoindol-2-yl)-acetic acidmethyl ester (19.85 mmol) in 20 ml of n-butanol was added and stirringwas continued at 95° C. to 100° C. for 1 h. Subsequently, the solventwas evaporated in vacuo, 25 ml of aqueous 2N HCl and 100 ml of ethylacetate were added and the mixture was stirred vigorously for 1 h beforeit was filtered by suction. The filter cake was washed thoroughly withwater, and dried in vacuo at 60° C. to give 4.43 g of a yellow solid.4.4 g of this mixture of A and B were separated by flash columnchromatography on silica gel eluting with dichloromethane:ethyl acetate(98:2). Evaporation of the first fraction yielded 1.99 g of A; ¹H NMR(CDCl₃): δ=10.48 (bs, 1H), 8.39 (bs, 1H), 8.24 (d, 1H), 8.01 (d, 1H),7.35 to 7.55 (m, 6H), 4.39 (t, 2H), 1.77 (m, 2H), 1.46 (m, 2H), 0.99 (t,3H). Evaporation of the second fraction yielded 2.26 g of B; ¹H NMR(CDCl₃): δ=10.38 (bs, 1H), 8.32 (bs, 1H), 8.24 (d, 1H), 7.86 (d, 1H),7.37 to 7.56 (m, 6H), 4.39 (t, 2H), 1.77 (m, 2H), 1.46 (m, 2H), 0.99 (t,3H).

e) 1-Bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acidbutyl ester

To a solution of 4.59 g of phosphorous oxybromide (16 mmol) in 25 ml ofanhydrous acetonitrile were added 1.108 g of1,4-dihydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butylester (3 mmol) and the mixture was refluxed gently with stirring for 1h. Then 5.04 g of sodium bicarbonate (60 mmol) were added, followed bythe dropwise addition of 8 ml of water. After stirring at ambienttemperature for 90 min the mixture was concentrated in vacuo to aboutone third of its volume, 40 ml of water were added and the mixture wasextracted with 30 ml of ethyl acetate. The mixture was filtered bysuction. The organic phase was separated, dried over MgSO₄, and filteredthrough a pad of silica gel. Evaporation in vacuo gave 0.885 g of thetitle compound; ¹H NMR (CDCl₃): δ=11.84 (s, 1H), 8.21 (d, 1H), 7.91 (d,1H), 7.40 to 7.55 (m, 6H), 4.46 (t, 2H), 1.84 (m, 2H), 1.48 (m, 2H),0.98 (t, 3H).

f) 4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butyl ester

A mixture of 432 mg of1-bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butylester (1 mmol), 63 mg of red phosphorous (2 mmol), 0.4 ml of aqueous 57wt % HI (3 mmol), and 1 ml of glacial acetic acid was refluxed withstirring for 30 min. Then the reaction mixture was diluted with 25 ml ofethyl acetate, filtered by suction through a pad of celite, washed witha solution of 0.2 g of NaHSO₃ in 5 ml of water, and washed twice with 5ml of concentrated aqueous sodium bicarbonate solution. The organicphase was dried over MgSO₄ and evaporated in vacuo. The residue waspurified by flash column chromatography on silica gel eluting withhexanes:ethyl acetate (85:15). 123 mg of the title compound wereobtained; ¹H NMR (CDCl₃): δ=11.85 (s, 1H), 8.60 (s, 1H), 8.23 (d, 1H),7.38 to 7.63 (m, 7H), 4.49 (t, 2 H), 1.87 (m, 2H), 1.47 (m, 2H), 0.98(t, 3H).

g) [(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid

A mixture of 113 mg of4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butyl ester(0.32 mmol), 244 mg of glycine (3.2 mmol), and 6.4 ml of a 0.5 Nsolution of sodium methoxide in methanol (3.2 mmol) was refluxed for 24h with stirring. Then the solvent was evaporated in vacuo, the residuedissolved in 25 ml of water and the resulting solution was washed twicewith 50 ml of ethyl acetate. The pH of the solution was subsequentlyadjusted to about 3 by addition of concentrated hydrochloric acid andthe resulting slurry was extracted twice with 25 ml of ethyl acetate.The combined extracts were dried over MgSO₄ and evaporated in vacuo. 103mg of the title compound were obtained; ¹H NMR (DMSO-d₆): δ=9.32 (t,1H), 8.74 (s, 1H), 8.19 (d, 1H), 7.94 (d, 1H), 7.45 to 7.65 (m, 6H),4.02 (d, 2H).

Example D-2[(4-Hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acida) 1-Bromo-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carboxylic acidbutyl ester

1.447 g of compound B (4 mmol) from Example D-1d) were reacted withphosphorous oxybromide analogously to Example D-1 e). The crude productwas purified by flash column chromatography on silica gel eluting withdichloromethane. 0.985 g of the title compound were obtained byevaporation of the first fraction; ¹H NMR (CDCl₃): δ=11.77 (s, 1H), 8.08(d, 1H), 8.05 (s, 1H), 7.41 to 7.56 (m, 6H), 4.46 (t, 2H), 1.85 (m, 2H),1.48 (m, 2H), 0.98 (t, 3H).

b) 4-Hydroxy-6-phenylsulfanyl-isoquinoline-3-carboxylic acid butyl ester

540 mg of 1-bromo-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester (1.25 mmol) were reacted with red phosphorous and HIanalogously to Example D-1 f). The crude product was purified by flashcolumn chromatography on silica gel eluting with hexanes:ethyl acetate(85:15). 150 mg of the title compound were obtained; ¹H NMR (CDCl₃):δ=11.78 (s, 1H), 8.71 (d, 1H), 8.11 (t, 1H), 7.79 (d, 1H), 7.39 to 7.54(m, 6H), 4.49 (t, 2H), 1.87 (m, 2H), 1.47 (m, 2H), 0.98 (t, 3H).

c) [(4-Hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid

127 mg of 4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carboxylic acidbutyl ester (0.36 mmol) were reacted with glycine and sodium methylateanalogously to Example D-1 g). 118 mg of the title compound wereobtained; ¹H NMR (DMSO-d₆): δ=9.33 (t, 1H), 8.80 (s, 1H), 8.11 (d, 1H),7.79 (s, 1H), 7.49 to 7.65 (m, 6H), 4.01 (d, 2H).

Example D-3[(1-Chloro-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid a. 1-Chloro-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester

A mixture of 554 mg of compound A (1.5 mmol) from Example D-1d) and 5 mlof phosphorous oxychloride was refluxed gently with stirring for 30 min.Then the excess phosphorous oxychloride was evaporated in vacuo, and theresidue was dissolved in 15 ml of acetonitrile. 2.94 g of sodiumbicarbonate (35 mmol) was added, followed by the dropwise addition of 4ml of water. After stirring for 1 h the mixture was concentrated invacuo to about one third of its volume, 20 ml of water were added andthe mixture was extracted twice with 20 ml of ethyl acetate. Thecombined organic phases were dried over MgSO₄ and filtered through a padof silica gel by suction. Evaporation in vacuo gave 426 mg of the titlecompound; ¹H NMR (CDCl₃): δ=11.85 (s, 1H), 8.23 (d, 1H), 7.95 (d, 1H),7.50 to 7.57 (m, 6H), 4.47 (t, 2H), 1.84 (m, 2H), 1.48 (m, 2H), 0.98 (t,3H).

b)[(1-Chloro-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid

194 mg of 1-chloro-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester (0.5 mmol) were reacted with glycine and sodiummethylate analogously to Example D-1 g). 168 mg of the title compoundwere obtained; ¹H NMR (DMSO-d₆): δ=9.17 (t, 1H), 8.24 (d, 1H), 7.51 to7.79 (m, 7H), 4.00 (d, 2H).

Example D-4[(1-Chloro-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid a) 1-Chloro-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester

554 mg of compound B (1.5 mmol) from Example D-1d) were reacted withphosphorous oxychloride analogously to Example D-3a). The crude productwas purified by flash column chromatography on silica gel eluting withdichloromethane. 205 mg of the title compound were obtained byevaporation of the first fraction; ¹H NMR (CDCl₃): δ=11.78 (s, 1H), 8.08(d, 1H), 8.06 (s, 1H), 7.41 to 7.56 (m, 6H), 4.46 (t, 2H), 1.85 (m, 2H),1.48 (m, 2H), 0.98 (t, 3H).

b)[(1-Chloro-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid

194 mg of 1-chloro-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester (0.5 mmol) were reacted with glycine and sodiummethylate analogously to Example D-1 g). 155 mg of the title compoundwere obtained; ¹H NMR (DMSO-d₆): δ=9.19 (t, 1H), 8.18 (d, 1H), 7.52 to7.79 (m, 7H), 4.00 (d, 2H).

Example D-5[(1-Bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid

216 mg of 1-bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester (0.5 mmol) from Example D-1 e) were reacted withglycine and sodium methylate analogously to Example D-1 g). 192 mg ofthe title compound were obtained; ¹H NMR (DMSO-d₆): δ=9.15 (t, 1H), 8.22(d, 1H), 7.52 to 7.74 (m, 7H), 4.01 (d, 2H).

Example D-6[(1-Bromo-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid

216 mg of 1-bromo-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester (0.5 mmol) from Example D-2a) were reacted with glycineand sodium methylate analogously to Example D-1 g). 194 mg of the titlecompound were obtained; ¹H NMR (DMSO-d₆): δ=9.17 (t, 1H), 8.12 (d, 1H),7.51 to 7.78 (m, 7H), 4.00 (d, 2H).

Example D-7 [(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 4-Phenoxy-phthalic acid

Synthesized from 4-phenoxy-phthalonitrile in analogy to Example D-1 a);MS-(−)-ion: M−1=256.9.

b) (1,3-Dioxo-5-phenoxy-1,3-dihydro-isoindol-2-yl)-acetic acid

Synthesized from 4-phenoxy-phthalic acid in analogy to Example D-1 b);MS-(+)-ion: M+1=297.9.

c) (1,3-Dioxo-5-phenoxy-1,3-dihydro-isoindol-2-yl)-acetic acid methylester

Synthesized from (1,3-dioxo-5-phenoxy-1,3-dihydro-isoindol-2-yl)-aceticacid in analogy to Example D-1c) (purification of the crude product byflash column chromatography on silica gel eluting with hexanes:ethylacetate (1:1)); ¹H NMR (CDCl₃): δ=7.83 (d, 1H), 7.05 to 7.46 (m, 7H),4.41 (s, 2H), 3.76 (s, 3H).

d) 1,4-Dihydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butyl ester(A) and 1,4-Dihydroxy-6-phenoxy-isoquinoline-3-carboxylic acid butylester (B)

Synthesized from (1,3-dioxo-5-phenoxy-1,3-dihydro-isoindol-2-yl)-aceticacid methyl ester in analogy to Example D-1 d); A: ¹H NMR (CDCl₃):δ=10.58 (bs, 1H), 8.37 (bs, 1H), 8.14 (d, 1H), 7.87 (d, 1H), 7.05 to7.49 (m, 6H), 4.39 (t, 2H), 1.77 (m, 2H), 1.46 (m, 2H), 0.99 (t, 3H); B:¹H NMR (CDCl₃): δ=10.38 (bs, 1H), 8.38 (d, 1H), 8.28 (bs, 1H), 7.56 (d,1 H), 7.06 to 7.47 (m, 6H), 4.40 (t, 2H), 1.77 (m, 2H), 1.46 (m, 2H),0.99 (t, 3H).

e) 1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butylester

Synthesized from 1,4-dihydroxy-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester in analogy to Example D-1e); ¹H NMR (CDCl₃): δ=11.89 (s,1H), 8.35 (d, 1H), 7.63 (d, 1H), 7.08 to 7.52 (m, 6H), 4.47 (t, 2H),1.84 (m, 2H), 1.48 (m, 2H), 0.99 (t, 3H).

f) 4-Hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butyl ester

A mixture of 208 mg of1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butyl ester(0.5 mmol), 49 mg of sodium acetate (0.6 mmol), 50 mg of 10 wt %palladium on charcoal, 10 ml of methanol, and 5 ml of ethyl acetate wasstirred under hydrogen at 1 atm for 15 h. Then the mixture was filteredby suction through a pad of celite and was concentrated in vacuo. Theresidue was partitioned between 2 ml of half concentrated aqueousbicarbonate solution and 8 ml of ethyl acetate. The organic phase wasdried over MgSO₄. Evaporation in vacuo gave 130 mg of the titlecompound; ¹H NMR (CDCl₃): δ=11.89 (bs, 1H), 8.61 (s, 1H), 8.36 (d, 1H),7.10 to 7.53 (m, 7H), 4.49 (t, 2H), 1.87 (m, 2H), 1.47 (m, 2H), 0.98 (t,3H).

g) [(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid

Synthesized from 4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester in analogy to Example D-1 g); ¹H NMR (DMSO-d₆): δ=9.29 (t,1H), 8.75 (s, 1H), 8.28 (d, 1H), 7.18 to 7.63 (m, 6H), 4.01 (d, 2H).

Alternatively, the title compound is prepared as follows:

a′) 4-Bromo-2-methyl-benzoic acid ethyl ester

25.3 g of 4-bromo-2-methyl benzoic acid and 5 mL of concentratedsulfuric acid were added to 425 mL of ethanol. The mixture was heated atreflux temperature for 3 days. The solution was cooled to roomtemperature, adjusted to neutral pH with the addition of sodiumbicarbonate, and concentrated to ca. 100 mL volume under reducedpressure. The reduced mixture was partitioned between ethyl acetate andwater, and the organic phase was successively washed with saturatedbicarbonate and brine solutions. The organic fraction was dried overanhydrous sodium sulfate, and concentrated to 28.2 g of a clear liquidproduct; ¹H NMR (200 MHz, CDCl₃) δ 7.78-7.73 (d, J=8.2 Hz, 1H),7.38-7.32 (m, 2H), 4.40-4.28 (q, J=7 Hz, 2H), 2.57 (s, 3H), 1.42-1.35(t, J=7 Hz, 3H).

b′) 2-Methyl-4-phenoxy-benzoic acid ethyl ester

27.5 g of 4-bromo-2-methyl-benzoic acid ethyl ester was dissolved in 120mL of anhydrous toluene. To the solution was added 21.3 g of phenol,73.6 g of Cs₂CO₃, 551 μL of ethyl acetate, 22 g of activated 4 Amolecular sieves, and 5.68 g of 90% copper(I) trifluoromethanesulfonatebenzene complex. The reaction was placed under a nitrogen atmosphere andheated at reflux temperature for 48 h. The resultant mixture waspartitioned between water and ethyl acetate, and the mixture filteredthrough a fine sintered glass filter to remove insoluble material. Theorganic fraction was washed three times with 1.0 N NaOH, once withbrine, dried over anhydrous sodium sulfate, and concentrated to 18.2 gof a pale tan liquid: ¹H NMR (200 MHz, CDCl₃) δ 7.93-7.88 (dd, J=1.3,7.8 Hz, 1H) 7.39-7.30 (m, 2H), 7.19-7.10 (tt, J=1.2, 7.4 Hz, 1H),7.06-7.7.0 (m, 2H), 6.80-6.75 (m, 2H), 4.37-4.26 (q, J=7.0 Hz, 2H), 2.56(s, 3H), 1.40-1.33 (t, J=7.0 Hz, 3H).

c′)2-{[(2,4-Dimethoxy-benzyl)-ethoxycarbonylmethyl-amino]-methyl}-4-phenoxy-benzoicacid ethyl ester

Ethyl N-(2,4-dimethoxybenzyl)glycinate was prepared following literatureprocedures. (Ananthan S, et al., J. Med. Chem. (1993), 36(4), pp479-490.) 13.0 g of 2-Methyl-4-phenoxy-benzoic acid ethyl ester wasdissolved in 102 mL of carbon tetrachloride. To the solution was added9.05 g of N-bromosuccinamide and 492 mg of benzoyl peroxide. The mixturewas heated at reflux temperature for 18 h under a nitrogen atmosphere,cooled to room temperature, and filtered through a pad of silica gel toremove all insoluble material. The resultant solution was concentratedto 16.5 g of a crude oil.

2.0 g of the above crude oil was dissolved in 10 mL of anhydrous DMF. Tothe solution was added 1.0 g of Ethyl N-(2,4-dimethoxybenzyl)glicinateand 552 mg of potassium carbonate. The reaction mixture was stirred for16 h. under a nitrogen atmosphere. The resultant mixture was poured into80 mL of water, and extracted three times with 50 mL portions of ethylacetate. The combined organic fractions were washed successively withhalf-saturated bicarbonate solution and brine. The organic fractionswere concentrated to an oily residue under reduced pressure, andre-suspended in 50 mL of ether and 10 mL hexanes. The solution wascooled to 0 deg C., and filtered to remove trace insoluble material. Asolution of 4 M HCl in dioxane was added slowly to the cold solution toprecipitate out solid material. The solid salt was collected byfiltration, and washed twice with cold ether. The solid was thendissolved by partitioning between 150 mL of ethyl acetate and 100 mL ofaqueous sodium bicarbonate solution. The organic fraction was separated,washed with brine, dried over anhydrous sodium sulfate, and concentratedto provide 1.8 g of a tan oil; MS (+) m/z 508.13 (m+1).

d′)2-(2,4-Dimethoxy-benzyl)-4-hydroxy-7-phenoxy-1,2-dihydro-isoquinoline-3-carboxylicacid ethyl ester

460 mg of2-{[(2,4-Dimethoxy-benzyl)-ethoxycarbonylmethyl-amino]-methyl}-4-phenoxy-benzoicacid ethyl ester was dissolved in 16 mL of anhydrous THF and theresultant solution cooled to −78 deg C. under a nitrogen atmosphere. Tothe solution was added 1.95 mL of 1.0 M lithium bis(trimethylsilyl)amide in THF. The reaction was stirred at −78 deg C. for 1.5 h, and atroom temperature for 4.5 hours. The resultant solution was poured into asolution of saturated aqueous ammonium chloride and extracted threetimes with ethyl acetate. The organic fractions were washed with brine,dried over anhydrous sodium sulfate and concentrated to yellow oil. Theoil was flash columned on silica gel, eluting with a gradient of 20-75%ethyl acetate in hexanes. The eluted fractions were concentrated underreduced pressure to 373 mg of a yellow oil, which was determined to be amixture of the enol and keto tautomers of the desired product; MS (+)m/z 484.20 (m+23).

e′) 4-Hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid ethyl ester

365 mg of2-(2,4-dimethoxy-benzyl)-4-hydroxy-7-phenoxy-1,2-dihydro-isoquinoline-3-carboxylicacid ethyl ester was dissolved in 7.9 ml of dichloromethane. To thesolution was added 92 μL of thionyl chloride. The reaction was stirredat room temperature for 6.5 h, and then 500 μL of ethanol was added andthe reaction stirred for an additional 10 min. The mixture waspartitioned between ethyl acetate and sodium bicarbonate. The organicfraction was successively washed with 0.5 M HCl, water, brine; driedover anhydrous sodium sulfate, and concentrated to 468 mg of a yellowoil. The oil was purified by flash chromatography on silica gel, elutingwith a gradient of 15-50% ethyl acetate in hexanes, to produce 232 mg ofcrude product, which was crystallized from ether and hexanes to give 193mg of off-white solid; MS (+) m/z 310.08 (m+1).

f′) [(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid

The title compound is prepared from4-Hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid ethyl ester underconditions analogous to example D1-g.

Another alternative synthetic route is for4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid ethyl ester providedbelow.

a″) 2-Dibromomethyl-4-phenoxy-benzoic acid ethyl ester

To a flask with 2-methyl-4-phenoxy-benzoic acid ethyl ester (example D-7b′), 3.05 g), N-bromosuccinamide (4.65 g), and benzylperoxide (115 mg)was added carbon tetrachloride 40 mL. The resulting mixture was refluxedfor 16 h under nitrogen. The insoluble was filtered off andconcentrated. The oil was diluted with 10% ethyl acetate in hexanes (50mL) and filtered through a pad of silica gel, further rinse with thesame solvent mix was continued twice. The filtrate solution wasconcentrated to give 5 g of 2-dibromomethyl-4-phenoxy-benzoic acid ethylester as an oil. ¹H NMR (200 MHz, CDCl₃) δ 8.07 (s, 1H), 7.86 (d, J=9Hz, 1H), 7.74 (d, J=2.4 Hz, 1H), 7.41 (d, J=7.6, 2H), 7.21 (m, 1H), 7.08(m, 2H), 6.86 (dd, J=2.5, 8.7, 1H), 4.37 (q, J=7.0 Hz, 2H), 1.40 (t,J=7.0 Hz, 3H).

b″) 2-Formyl-4-phenoxy-benzoic acid ethyl ester

2-Dibromomethyl-4-phenoxy-benzoic acid ethyl ester (2.07 g) wasdissolved in tetrahydrofuran (40 mL) and water (15 mL). Silver nitrate(2.56 g) was added. The resulting mixture was heated to reflux for 5 h.The precipitate was filtered off and the reaction was diluted with ethylacetate. The organic layer was separated and the aqueous layer wasextracted again with ethyl acetate. The combined ethyl acetate layer waswashed with saturated sodium bicarbonate solution, brine, and dried withmagnesium sulfate. After concentration, the crude oil was diluted with20% ethyl acetate in hexanes (100 mL) and filtered through a pad ofsilica gel. Further rinse was continued twice. The filtrate solution wasconcentrated to give the title compound 1.13 g as an oil. ¹H NMR (200MHz, CDCl₃) δ 10.62 (s, 1H), 7.98 (d, J=8.6 Hz, 1H), 7.42-7.35 (m, 3H),7.20-7.14 (m, 2H), 7.04 (d, J=8.2, 2H), 4.41 (q, J=7.0 Hz, 2H), 1.41 (t,J=7.0 Hz, 3H).

c″) 2-(Ethoxycarbonylmethylimino-methyl)-4-phenoxy-benzoic acid ethylester

To a dried flask with glycine ethyl ester hydrochloride salt (62 mg) wasadded anhydrous dichloromethane (2 mL), followed by triethylamine (124μL). Then magnesium sulfate (pre-dried on high vacuum by heat gun, 100mg) was added, followed by addition of a dichloromethane (1 mL) solutionof 2-formyl-4-phenoxy-benzoic acid ethyl ester (120 mg). The flask of2-formyl-4-phenoxy-benzoic acid ethyl ester was further rinsed with 0.5mL of dichloromethane. The resulting mixture was stirred at roomtemperature under nitrogen for 15 h. The mixture was filtered and rinsedwith dichloromethane. After removal of solvent, the reaction was dilutedwith ether (15 mL) and washed with brine twice and dried. Filtration andremoval of solvent gave the title compound 160 mg as an oil with goodpurity. ¹H NMR (200 MHz, CDCl₃) δ 9.02 (d, J=1.2, 1H), 7.94 (d, J=8.6Hz, 1H), 7.63 (d, J=2.4, 1H), 7.40-7.32 (m, 2H), 7.20-7.11 (m, 1H),7.06-6.97 (m, 3H), 4.41 (s, 2H), 4.35 (q, J=7.0 Hz, 2H), 4.21 (q, J=7.4Hz, 2H), 1.39 (t, J=7.0 Hz, 3H), 1.28 (t, J=7.0 Hz, 3H).

d″) 4-Hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid ethyl ester

Potassium tert-butoxide (47 mg) was dried on high-vacuum pump around 90degree for more than one hour. Under nitrogen, anhydrous tetrahydrofuran(1.4 mL) was added to it followed by a tetrahydrofuran solution (1.6 mL)of 2 (ethoxycarbonylmethylimino-methyl)-4-phenoxy-benzoic acid ethylester (60 mg) and a further 0.5 mL of tetrahydrofuran. The mixtureturned orange-red. After stirring at room temperature for 2.5 h, themixture was refluxed for another 2.5 h and then quenched with water (5mL). Ethyl acetate (30 mL) was added. The organic phase was separatedand washed with brine and dried. Removal of solvent gave 26 mg of thetitle compound as an oil with good purity. ¹H NMR (200 MHz, CDCl₃):identical to that of example D-7 e′).

Example D-8 [(4-Hydroxy-6-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 1-Bromo-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylic acid butylester

Synthesized from dihydroxy-6-phenoxy-isoquinoline-3-carboxylic acidbutyl ester from Example D-7 d) in analogy to Example D-1e); ¹H NMR(CDCl₃): δ=11.76 (s, 1H), 8.22 (d, 1 H), 7.68 (d, 1H), 7.10 to 7.55 (m,6H), 4.46 (t, 2H), 1.85 (m, 2H), 1.48 (m, 2H), 0.99 (t, 3H).

b) 4-Hydroxy-6-phenoxy-isoquinoline-3-carboxylic acid butyl ester

Synthesized from 1-bromo-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylicacid butyl ester in analogy to Example D-7 f); ¹H NMR (CDCl₃): δ=11.76(s, 1H), 8.74 (s, 1H), 7.93 (d, 1H), 7.69 (d, 1H), 7.10 to 7.52 (m, 6H),4.49 (t, 2H), 1.87 (m, 2H), 1.47 (m, 2H), 0.98 (t, 3H).

c) [(4-Hydroxy-6-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid

Synthesized from 4-hydroxy-6-phenoxy-isoquinoline-3-carboxylic acidbutyl ester in analogy to Example D-1g); ¹H NMR (DMSO-d₆): δ=9.33 (t,1H), 8.82 (s, 1H), 8.23 (d, 1H), 7.20 to 7.63 (m, 7H), 4.01 (d, 2H).

Example D-9[(1-Chloro-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 1-Chloro-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from 1,4-dihydroxy-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester from Example D-7d) in analogy to Example D-3a); ¹H NMR(CDCl₃): δ=11.90 (s, 1H), 8.37 (d, 1H), 7.10 to 7.64 (m, 7H), 4.47 (t,2H), 1.84 (m, 2H), 1.48 (m, 2H), 0.99 (t, 3H).

b) [(1-Chloro-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from 1-chloro-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylicacid butyl ester in analogy to Example D-1g); ¹H NMR (DMSO-D₆): δ=9.16(t, 1H), 8.36 (d, 1H), 7.23 to 7.72 (m, 7H), 4.01 (d, 2H).

Example D-10[(1-Chloro-4-hydroxy-6-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 1-Chloro-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from 1,4-dihydroxy-6-phenoxy-isoquinoline-3-carboxylic acidbutyl ester from Example D-7d) in analogy to Example D-3a); ¹H NMR(CDCl₃): δ=11.77 (s, 1H), 8.25 (d, 1H), 7.69 (d, 1H), 7.10 to 7.55 (m,6H), 4.47 (t, 2H), 1.85 (m, 2H), 1.48 (m, 2H), 0.98 (t, 3H).

b) [(1-Chloro-4-hydroxy-6-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from 1-chloro-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylicacid butyl ester in analogy to Example D-1g); ¹H NMR (DMSO-d₆): δ=9.19(t, 1H), 8.31 (d, 1H), 7.23 to 7.74 (m, 7H), 4.00 (d, 2H).

Example D-11[(1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from 1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylicacid butyl ester from Example D-7 e) in analogy to Example D-1g);MS-(+)-ion: M+1=417.0

Example D-12[(1-Bromo-4-hydroxy-6-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from 1-bromo-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylicacid butyl ester from Example D-8 a) in analogy to Example D-1g);MS-(−)-ion: M−1=414.9.

Example D-13{[7-(2,6-Dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-(2,6-Dimethyl-phenoxy)-phthalic acid

Synthesized from 4-(2,6-dimethyl-phenoxy)-phthalonitrile in analogy toExample D-1 a); ¹H NMR (CDCl₃): δ=7.89 (d, 1H), 7.19 (d, 1H), 7.08 (bs,3H), 6.79 (m, 1H), 2.10 (s, 6H).

b) [5-(2,6-Dimethyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

4-(2,6-dimethyl-phenoxy)-phthalic acid was reacted with glycine inanalogy to Example D-1 b). The crude product was then reacted withmethanol in analogy to Example D-1 c); ¹H NMR (CDCl₃): δ=7.80 (d, 1H),7.09 to 7.17 (m, 5H), 4.40 (s, 2H), 3.76 (s, 3H), 2.11 (s, 6H).

c) 7-(2,6-Dimethyl-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester

0.79 g of sodium (34 mmol) were dissolved in 100 ml of n-butanol withstirring. Then the temperature was raised to 95° C. to 100° C., 5.70 gof [5-(2,6-dimethyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester (16.8 mmol) were added in one portion and stirring wascontinued at 95° C. to 100° C. for 3 h. Subsequently, the solvent wasevaporated in vacuo, 25 ml of aqueous 2N HCl and 100 ml of ethyl acetatewere added and the mixture was stirred vigorously for 30 min before itwas filtered by suction. The organic phase was separated from thefiltrate, dried over MgSO₄ and was evaporated in vacuo to give a browngum that was triturated with methanol. The resulting precipitate wasfiltered by suction and dried in vacuo to give 870 mg of a yellowishsolid (A). The filtrate was evaporated in vacuo, dissolved in a smallamount of methanol and stored overnight in a refrigerator. The resultingprecipitate was filtered by suction and dried in vacuo to give 246 mg ofa yellowish solid (B). A and B were pooled and purified by flash columnchromatography on silica gel eluting with dichloromethane:ethyl acetate(98:2). Evaporation of the first fraction yielded 762 mg of the titlecompound; ¹H NMR (CDCl₃): δ=8.31 (bs, 1H), 8.12 (d, 1H), 7.60 (d, 1H),7.35 (m, 1H), 7.09 (bs, 3H), 4.39 (t, 2 H), 2.11 (s, 6H), 1.77 (m, 2H),1.44 (m, 2H), 0.99 (t, 3H).

d) 1-Bromo-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Synthesized from7-(2,6-dimethyl-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester in analogy to Example D-1 e); ¹H NMR (CDCl₃): δ=11.88 (s,1H), 8.33 (m, 1 H), 7.35 to 7.40 (m, 2H), 7.13 to 7.16 (m, 3H), 4.46 (t,2H), 2.14 (s, 6H), 1.83 (m, 2H), 1.48 (m, 2H), 0.98 (t, 3H).

e) 7-(2,6-Dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from1-bromo-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester in analogy to Example D-7 f); ¹H NMR (CDCl₃): δ=11.87(s, 1H), 8.35 (s, 1H), 8.36 (d, 1H), 7.47 (dd, 1H), 7.14 (m, 2H), 6.87(d, 1H), 4.48 (t, 2H), 2.14 (s, 6 H), 1.87 (m, 2H), 1.47 (m, 2H), 0.98(t, 3H).

f){[7-(2,6-Dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Synthesized from7-(2,6-Dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester in analogy to Example D-1 g); MS-(+)-ion: M+1=367.1.

Example D-14{[1-Chloro-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]amino}-aceticacid a)1-Chloro-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Synthesized from7-(2,6-dimethyl-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester from Example D-13 c) in analogy to Example D-3 a); ¹H NMR(CDCl₃): δ=11.89 (s, 1H), 8.35 (d, 1H), 7.34 to 7.43 (m, 2H), 7.13 to7.14 (m, 3H), 4.47 (t, 2H), 2.14 (s, 6 H), 1.85 (m, 2H), 1.48 (m, 2H),0.99 (t, 3H).

b){[1-Chloro-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Synthesized from1-chloro-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester in analogy to Example D-1 g); MS-(−)-ion: M−1=398.9.

Example D-15

{[1-Bromo-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Synthesized from1-bromo-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester from Example D-13 d) in analogy to Example D-1 g);MS-(−)-ion: M-1=442.9.

Example D-16[(1-Bromo-7-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acida) (5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid

Synthesized in analogy to Example D-1 b)(5-chloro-isobenzofuran-1,3-dione was used as starting material insteadof the corresponding phthalic acid); ¹H NMR (DMSO-d₆/D₂O): δ=8.01 (s,1H), 7.93 (s, 2H), 4.32 (s, 2H).

b) (5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid methylester

Synthesized from (5-chloro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-aceticacid in analogy to Example D-1 c); ¹H NMR (CDCl₃): δ=7.67 to 7.86 (m,3H), 4.43 (s, 2H), 3.76 (s, 3H).

c) 7-Chloro-1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester (A)and 6-Chloro-1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester(B)

Synthesized from (5-chloro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-aceticacid methyl ester in analogy to Example D-1 d) (pure B was obtained byrecrystallization from chloroform after chromatography); A: ¹H NMR(CDCl₃): δ=8.46 (bs, 1H), 8.41 (d, 1H), 8.10 (d, 1H), 7.73 (dd, 1H),4.41 (t, 2H), 1.77 (m, 2H), 1.46 (m, 2H), 1.00 (t, 3H); B: ¹H NMR(CDCl₃): δ=8.34 to 8.38 (m, 2H), 8.12 (d, 1H), 7.64 (dd, 1H), 4.42 (t,2H), 1.77 (m, 2H), 1.46 (m, 2H), 1.00 (t, 3H).

d) 1-Bromo-7-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

Synthesized from 7-Chloro-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester in analogy to Example D-1 e); ¹H NMR (CDCl₃): δ=11.92 (s,1H), 8.34 (d, 1H), 8.25 (d, 1H), 7.75 (dd, 1H), 4.49 (t, 2H), 1.86 (m,2H), 1.48 (m, 2H), 1.00 (t, 3H).

e) [(1-Bromo-7-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from 1-bromo-7-chloro-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester in analogy to Example D-1 g); MS-(−)-ion: M−1=356.8.

Example D-17[(1-Bromo-6-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acida) 1-Bromo-6-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

Synthesized from 6-chloro-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester from Example D-16 c) in analogy to Example D-1 e); ¹H NMR(CDCl₃): δ=11.88 (s, 1H), 8.37 (d, 1H), 8.19 (d, 1H), 7.75 (dd, 1H),4.49 (t, 2H), 1.86 (m, 2H), 1.48 (m, 2H), 0.99 (t, 3H).

b) [(1-Bromo-6-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from 1-bromo-6-chloro-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester in analogy to Example D-1 g); MS-(−)-ion: M−1=356.9.

Example D-18[(1-Bromo-4-hydroxy-7-trifluoromethyl-isoquinoline-3-carbonyl)-amino]-aceticacid a) (1,3-Dioxo-5-trifluoromethyl-1,3-dihydro-isoindol-2-yl)-aceticacid methyl ester

4-trifluoromethyl-phthalic acid was reacted with glycine in analogy toExample D-1 b). The crude product was then reacted with methanol inanalogy to Example D-1 c); ¹H NMR (CDCl₃): δ=8.14 (s, 1H), 8.02 (m, 2H),4.48 (s, 2H), 3.78 (s, 3H).

b) 1,4-Dihydroxy-7-trifluoromethyl-isoquinoline-3-carboxylic acid butylester (A) and 1,4-Dihydroxy-6-trifluoromethyl-isoquinoline-3-carboxylicacid butyl ester (B)

Synthesized from(1,3-dioxo-5-trifluoromethyl-1,3-dihydro-isoindol-2-yl)-acetic acidmethyl ester in analogy to Example D-1 d); A: ¹H NMR (CDCl₃): δ=10.47(bs, 1H), 8.76 (bs, 1 H), 8.72 (d, 1H), 8.29 (m, 1H), 7.99 (m, 1H), 4.45(t, 2H), 1.77 (m, 2H), 1.46 (m, 2H), 1.00 (t, 3H); B: ¹H NMR (CDCl₃):δ=10.48 (bs, 1H), 8.44 to 8.57 (m, 3H), 7.91 (d, 1H), 4.44 (t, 2 H),1.77 (m, 2H), 1.46 (m, 2H), 1.01 (t, 3H).

c) 1-Bromo-4-hydroxy-7-trifluoromethyl-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from1,4-dihydroxy-7-trifluoromethyl-isoquinoline-3-carboxylic acid butylester in analogy to Example D-1 e); ¹H NMR (CDCl₃): δ=11.96 (s, 1H),8.52 to 8.56 (m, 2H), 7.99 (dd, 1H), 4.51 (t, 2H), 1.86 (m, 2H), 1.48(m, 2H), 1.00 (t, 3H).

d)[(1-Bromo-4-hydroxy-7-trifluoromethyl-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from1-bromo-4-hydroxy-7-trifluoromethyl-isoquinoline-3-carboxylic acid butylester in analogy to Example D-1 g); MS-(−)-ion: M−1=391.0.

Example D-19[(1-Bromo-4-hydroxy-6-trifluoromethyl-isoquinoline-3-carbonyl)-amino]-aceticacid a) 1-Bromo-4-hydroxy-6-trifluoromethyl-isoquinoline-3-carboxylicacid butyl ester

Synthesized from1,4-dihydroxy-6-trifluoromethyl-isoquinoline-3-carboxylic acid butylester from Example D-18 b) in analogy to Example D-1 e); MS-(−)-ion:M−1=390.3.

b)[(1-Bromo-4-hydroxy-6-trifluoromethyl-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from1-bromo-4-hydroxy-6-trifluoromethyl-isoquinoline-3-carboxylic acid butylester in analogy to Example D-1 g); MS-(−)-ion: M−1=390.9.

Example D-20[(4-Hydroxy-1-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid a)(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid methyl ester

Synthesized from (1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid inanalogy to Example D-1 c); ¹H NMR (CDCl₃): δ=7.84 to 7.91 (m, 2H), 7.71to 7.77 (m, 2H), 4.45 (s, 2 H), 3.77 (s, 3H).

b) 1,4-Dihydroxy-isoquinoline-3-carboxylic acid butyl ester

Synthesized from (1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-acetic acidmethyl ester in analogy to Example D-1 d) (the solvent was notevaporated before adding hydrochloric acid, no ethyl acetate was added);¹H NMR (DMSO-d₆): δ=10.66 (bs, 1H), 10.55 (bs, 1H), 8.27 (d, 1 H), 8.08(d, 1H), 7.72 to 7.92 (m, 2H), 4.33 (t, 2H), 1.74 (m, 2H), 1.44 (m, 2H),0.93 (t, 3H).

c) 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

Synthesized from 1,4-Dihydroxy-isoquinoline-3-carboxylic acid butylester in analogy to Example D-3 a); ¹H NMR (CDCl₃): δ=11.91 (s, 1H),8.41 (m, 1H), 8.29 (m, 1H), 7.83 (m, 2H), 4.49 (t, 2H), 1.84 (m, 2H),1.48 (m, 2H), 0.99 (t, 3H).

d) 4-Hydroxy-1-phenoxy-isoquinoline-3-carboxylic acid butyl ester

A mixture of 1.399 g of 1-Chloro-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (5 mmol) and 2.86 g of phenol was heated at 145° C. to150° C. for 24 h. After cooling to ambient temperature the mixture wassuspended in 50 ml of aqueous 2N NaOH and the mixture was extracted with4×25 ml of ethyl acetate. The combined organic phases were washed with3×25 ml of aqueous 2N NaOH, 50 ml of brine, dried over MgSO₄, andevaporated in vacuo. The residue was purified by flash columnchromatography on silica gel eluting with hexanes:ethyl acetate (9:1)and (95:5). 0.650 g of the title compound were obtained; ¹H NMR (CDCl₃):δ=11.52 (s, 1H), 8.32 to 8.39 (m, 2H), 7.72 to 7.86 (m, 2H), 7.13 to7.42 (m, 5H), 4.31 (t, 2H), 1.69 (m, 2H), 1.37 (m, 2H), 0.93 (t, 3H).

e) [(4-Hydroxy-1-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid

Synthesized from 4-Hydroxy-1-phenoxy-isoquinoline-3-carboxylic acidbutyl ester in analogy to Example D-1 g); MS-(+)-ion: M+1=339.1.

Example D-21[(1,7-dibromo-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid a)(5-Bromo-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid ethyl ester

Bromophthalimide (35 g, 155 mmol) and bromoethylacetate (31 g, 186 mmol)were dissolved in 700 ml of acetone. Potassium carbonate (64.2 g, 465mmol) was added and resulting suspension was stirred at reflux for 18 h.After cooling, the mixture was filtered. Filtrate was evaporated to give48.12 g (154 mmol) of solid product. ¹H NMR (200 MHz, CDCl₃) δ 8.00 (s,1H), 7.89 (d, J=7.8 Hz, 1H), 7.73 (d, J=7.8 Hz, 1H), 4.41 (s, 2H), 4.21(q, J=7.0 Hz, 2H), 1.28 (t, J=7.0 Hz, 3H).

b) 6- and 7-Bromo-1,4-dihydroxy-isoquinoline-3-carboxylic acid butylester

Sodium (10.45 g) was dissolved in 460 ml of n-butanol with heating(45-50° C.). The above ester (68 g, 218 mmol) was dissolved in 460 ml ofn-butanol (heated to homogeneous), and then added to the sodiumsolution. Combined mixture was stirred mechanically at 75° C. for 1 h.Mixture was removed from heat and stirred at room temperature overnight.Solution was acidified using 2 N HCl to pH ˜3. Precipitate was collectedby vacuum filtration and washed with water and then methanol to give59.4 g (175 mmol) of product as a mixture of two isomers.

c) 7-Bromo-1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester

10 g of the above isomeric mixtures was subjected to silica gel flashchromatography eluting with 10% ethyl acetate in methylene chloride togive 3 g of product as white solid. MS-(+)-ion: M+1=342.02, 340.02

d) 1,7-dibromo-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

The above ester (2.4 g, 7.1 mmol) was dissolved in 150 ml of anhydrousacetonitrile. Phosphorous oxybromide (14.1 g, 49.4 mmol) was added.Mixture was stirred at reflux for 3 h. After cooling, the reactionmixture was concentrated and the residue was taken into ethyl acetate.Ethyl acetate mixture was poured into saturated sodium bicarbonatesolution with efficient stirring. Two phases were separated. Organiclayer was dried over magnesium sulfate, filtered and concentrated togive 2 g (5.0 mmol) of product. MS-(+)-ion: M+1=403.90

e) [(1,7-dibromo-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid

The above ester (0.2 g, 0.5 mmol) was dissolved in 5 ml of ethanol.Glycine (0.24 g, 9.9 mmol) and sodium ethoxide (0.34 g, 5 mmol) wereadded to the solution. Mixture was stirred at reflux for 3 days. Mixturewas evaporated. Residue was dissolved in water and washed with ethylacetate. Aqueous layer was acidified using 1N HCl aqueous solution topH=3-4, then extracted with ethyl acetate. Organic layer was dried overmagnesium sulfate, filtered and concentrated to give 0.17 g of productas white solid. ¹H NMR (200 MHz, DMSO-d6) δ 9.26 (t, J=6.2 Hz, 1H), 8.32(d, J=1.6 Hz, 1H), 8.23 (d, J=9.0 Hz, 1H), 8.11 (dd, J=9.0, 1.6 Hz, 1H), 4.02 (d, J=6.2 Hz, 2H).

Example D-22[(7-Bromo-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acida) 7-Bromo-1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

170 mg (0.5 mmol) of 7-bromo-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester (from Example D-21c) was dissolved in 2 ml of anhydrousacetonitrile. Phosphorous oxychloride (536 mg, 3.5 mmol) was added andthe resulting mixture was stirred at reflux for 4 h. After cooling, themixture was concentrated and the residue was taken into ethyl acetate.Ethyl acetate mixture was poured into saturated sodium bicarbonatesolution with efficient stirring for 1 h. Two phases were separated.Aqueous layer was extracted with ethyl acetate. Combined organic layerwas dried over magnesium sulfate, filtered and concentrated. Crudeproduct was purified by silica gel chromatography eluting with methylenechloride to give 78 mg of product as white solid. MS-(+)-ion:M+1=359.96, 357.98

b) [(7-Bromo-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

75 mg (0.21 mmol) of the above ester was reacted with glycine (314 mg,4.18 mmol) and sodium ethoxide (143 mg, 2.09 mmol) analogously toExample D-21e). 58 mg of product was obtained. ¹H NMR (200 MHz, CD₃OD) δ8.44 (d, J=1.6 Hz, 1H), 8.28 (d, J=9.0 Hz, 1H), 8.00 (dd, J=9.0, 1.6 Hz,1H), 4.17 (s, 2H).

Example D-23 [(6-Bromo-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 6-Bromo-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

2.58 g (6.40 mmol) of 6- and7-Bromo-1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl estermixtures (from Example D-21b) was dissolved in 30 ml of glacial aceticacid. A palladium (10% in activated carbon) slurry in 10 ml of glacialacetic acid was added. The mixture was stirred under hydrogen atmosphere(balloon pressure) for 2 h. Catalyst was filtered off through a pad ofcelite and rinsed with methylene chloride. Filtrated was concentratedand residue was triturated in methylene chloride. Insoluble solid wascollected by filtration and subjected to silica gel chromatographyeluting with (3/1) hexanes/ethyl acetate to give 192 mg of product.MS-(−)-ion: M−1=324.11, 322.13

b) [(6-Bromo-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid

178 mg (0.55 mmol) of the above ester was reacted with glycine (1.23 g,16.43 mmol) and sodium ethoxide (746 mg, 10.96 mmol) analogously toExample D-21e. The product obtained was further triturated with 30 ml ofmethanol to give 58 mg of product. ¹H NMR (200 MHz, CD₃OD) δ 8.72 (s,1H), 8.46 (s, 1H), 7.98 (d, J=8.8 Hz), 7.86 (d, J=8.8 Hz, 1H), 4.14 (s,2H).

Example D-24[(1-Bromo-7-fluoro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acida) (5-Fluoro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid methylester

A solid mixture of 5-fluoro-isobenzofuran-1,3-dione (3.68 g, 22.15 mmol)and glycine (1.66 g, 22.15 mmol) was stirred at 200-220° C. for 5 min.After cooling, it was dissolved in 25 ml of acetone. Methyl sulfate(4.19 g, 33.23 mmol) and potassium carbonate (4.59 g, 33.23 mmol) wasadded. The mixture was stirred at reflux for 2 h. After cooling, it wasdiluted with 100 ml of ethyl acetate. Insoluble was filtered off andfiltrate was concentrated. Residue was taken into 200 ml of ethylacetate and washed with water and brine. Ethyl acetate layer was driedover sodium sulfate, filtered, and concentrated to give 5.1 g ofproduct. ¹H NMR (200 MHz, CDCl₃) δ 7.88 (dd, J=8.2, 4.3 Hz, 1H), 7.54(dd, J=6.8, 2.2 Hz, 1H), 7.40 (m, 1H), 4.44 (s, 2 H), 3.77 (s, 3H).

b) 7-Fluoro-1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester (A)and 6-Fluoro-1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester(B)

3.0 g (12.66 mmol) of the above ester was rearranged analogously toExample D-21b at 95-100° C. for 2 h to give 2.5 g of product as amixture of isomers. The isomeric mixtures were purified by silica gelchromatography eluting with 5-20% ethyl acetate in methylene chloride.The first fraction was concentrated and recrystallized from 60 ml ofethanol to give 268 mg of solid product (A). The second fraction wasconcentrated to give 313 mg of solid product (B). For product A:MS-(−)-ion: M−1=278.02; For product B: MS-(−)-ion: M−1=278.03.

Differentiation of the isomers A and B can be measured on the silica gelTLC plate with 10% ethyl acetate in methylene chloride: A: R_(f) about0.79; B: R_(f) about 0.53)

c) 1-Bromo-7-fluoro-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

250 mg (0.90 mmol) of the above ester (A) was brominated analogously toExample D-21d (10% methanol in methylene chloride was used instead ofethyl acetate) to give 156 mg of solid product. MS-(+)-ion: M+1=344.00,341.99

d) [(1-Bromo-7-fluoro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

60 mg (0.18 mmol) of the above ester was reacted with glycineanalogously to Example D-21e. (Reaction time was 48 h). 10% Methanol inmethylene chloride was used to extracted the product. The organic layerwas dried over magnesium sulfate, filtered, and concentrated to give 50mg of the product. MS-(−)-ion: M−1=343.02, 340.92; ¹H NMR (200 MHz,acetone-d₆) δ 13.56 (s, 1H), 8.81 (br s, 1H), 8.43 (dd, J=9.0, 5.4 Hz,1H), 7.79 (m, 2H), 4.29 (d, J=6.2 Hz, 2H).

Example D-25 [(7-Fluoro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

42 mg (0.12 mmol) of the above carboxylic acid was dissolved in 5 ml of(4/1) methanol/water. Sodium carbonate (13 mg, 0.12 mmol) and palladium(wet, 10% dry basis on activated carbon) (40 mg) were added. The mixturewas stirred under hydrogen atmosphere (balloon pressure) for 2 h.Catalyst was filtered off through a pad of celite, rinsed with 10 ml of(4/1) methanol/water and then 2 ml of water. Filtrate was concentratedto remove most methanol and acidified by 1 N HCl to pH=3-4. Precipitatewas collected by filtration and dried under high vacuum to give 14 mg ofproduct. MS-(−)-ion: M−1=262.99; ¹H NMR (200 MHz, CD₃OD) δ 8.69 (s, 1H),8.38 (dd, J=9.0, 5.5 Hz, 1H), 7.24 (dd, J=9.3, 2.4 Hz, 1H), 7.60 (m,1H), 4.14 (s, 2H).

Example D-26[(1-Chloro-7-fluoro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a. 1-Chloro-7-fluoro-4-hydroxy-isoquinoline-3-carboxylic acid butylester

135 mg (0.48 mmol) of 7-fluoro-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester (product A from Example D-24b) was dissolved in 3 ml ofanhydrous acetonitrile. Phosphorous oxychloride (1.24 g, 8.07 mmol) wasadded. The mixture was stirred at reflux for 6 h. After cooling, it wasconcentrated and suspended in 10 ml of saturated sodium bicarbonateaqueous solution. Stirred for 1 h and extracted with 5% methanol inmethylene chloride. Organic layer was washed with brine, dried overmagnesium sulfate, filtered, and concentrated. Crude residue waspurified by silica gel chromatography eluting with methylene chloride togive 58 mg of product. MS-(−)-ion: M−1=296.12

b. [(1-Chloro-7-fluoro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

55 mg (0.19 mmol) of the above ester was reacted with glycineanalogously to Example D-21e. After acidification, it was extracted with10% methanol in methylene chloride. Organic layer was washed with brine,dried over magnesium sulfate, filtered, and concentrated. The residuewas purified by preparative TLC on 10% methanol in methylene chloride togive 6 mg of product. ¹H NMR (200 MHz, acetone-d6) δ 13.59 (s, 1H), 8.90(br s, 1H), 8.47 (dd, J=9.0, 5.1 Hz, 1H), 7.94 (dd, J=9.7, 2.4 Hz, 1H),7.81 (m, 1H), 4.28 (d, J=6.2 Hz, 2H).

Example D-27[(Chloro-4-hydroxy-benzo[g]isoquinoline-3-carbonyl)-amino]-acetic acida) (1,3-Dioxo-1,3-dihydro-benzo[f]isoindol-2-yl)-acetic acid ethyl ester

2 g (10.1 mmol) Benzo[f] isoindole-1,3-dione was reacted withbromoacetic acid ethyl ester analogously to Example D-21a. The crudeproduct obtained was partitioned between ethyl acetate and water.Organic layer was washed with brine, dried over magnesium sulfate,filtered, and evaporated to give 2.68 g (9.5 mmol) of product. ¹H NMR(200 MHz, CDCl₃) δ 8.36 (s, 2 H), 8.05 (m, 2H), 7.68 (m, 2H), 4.49 (s,2H), 4.22 (q, J=7.0 Hz, 2H), 1.29 (t, 7.0 Hz, 3H).

b) 1,4-Dihydroxy-benzo[g]isoquinoline-3-carboxylic acid butyl ester

2.6 g (9.2 mmol) of the above isoindol ester was rearranged analogouslyto Example D-21b to give 1.23 g (3.9 mmol) of product. ¹H NMR (200 MHz,CDCl₃) δ 10.73 (br s, 1H), 9.00 (s, 1H), 8.68 (s, 1H), 8.24 (br s, 1H),8.06 (m, 2H), 7.68 (m, 2H), 4.24 (t, J=6.6 Hz, 2H), 1.80 (m, 2H), 1.47(m, 2H), 1.00 (t, J=7.4 Hz, 3H).

c) 1-Chloro-4-hydroxy-benzo[g]isoquinoline-3-carboxylic acid butyl ester

1 g (3.2 mmol) of the above ester was reacted with 5 ml of phosphorousoxychloride analogously to Example D-22a without using acetonitrile as aco-solvent to give 0.88 g (2.7 mmol) of product. ¹H NMR (200 MHz, CDCl₃)δ 12.24 (s, 1H), 8.97 (s, 1H), 8.85 (s, 1H), 8.12 (m, 2H), 7.70 (m, 2H),4.51 (t, J=7.0 Hz, 2H), 1.89 (m, 2H), 1.56 (m, 2H), 1.00 (t, J=7.2 Hz,3H).

d) [(Chloro-4-hydroxy-benzo[g] isoquinoline-3-carbonyl)-amino]-aceticacid

0.88 g (2.7 mmol) of the above ester was reacted with glycineanalogously to Example D-21e. The resulting precipitate afteracidification was collected by filtration and dried in high vacuum togive 0.30 g (0.9 mmol) of product. ¹H NMR (200 MHz, DMSO-d6) δ 9.34 (brs, 1H), 9.00 (s, 1H), 8.92 (s, 1H), 8.34 (m, 2H), 7.74 (m, 2H), 3.94 (d,J=5.4 Hz, 2H).

Example D-28 [(1-Bromo-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 1-Bromo-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

2 g (7.7 mmol) of 1,4-Dihydroxy-isoquinoline-3-carboxylic acid butylester (from Example D-20b) dissolved in 100 ml of acetonitrile. 15.4 g(53.6 mmol) of phosphorous oxybromide added to solution and mixturestirred at 80° C. for 64 h. 100 ml of water was added to mixture, andmixture was removed from heat. Mixture was partitioned between ethylacetate and water. Two phases were separated and the aqueous layer wasextracted with ethyl acetate. Organic layers were combined, washed withbrine, dried over magnesium sulfate, filtered, and evaporated. Residuewas purified by silica gel flash chromatography to give 0.1 g (0.3 mmol)of product. ¹H NMR (200 MHz, CDCl₃) δ 11.89 (s, 1H), 8.41 (m, 1H), 8.25(m, 1H), 7.84 (m, 2 H), 4.49 (t, J=7.0 Hz, 2H), 1.87 (m, 2H), 1.47 (m,2H), 1.00 (t, J=7.2 Hz, 3H).

b) [(1-Bromo-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid

2 g (0.3 mmol) of the above isoquinoline ester was reacted with glycineanalogously to Example D-21e to give 0.08 g (0.2 mmol) of product. ¹HNMR (200 MHz, CD₃OD) δ 8.94 (br s, 1H), 8.34 (m, 1H), 8.24 (m, 1H), 7.86(m, 2H), 4.18 (d, J=6.2 Hz, 2H).

Example D-29 [(4-Hydroxy-6-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was prepared from[(1-Chloro-4-hydroxy-6-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid, Example D-33, following a procedure analogous to that described indetail in Example D-37. The final product was purified by chromatographyon silica gel using a gradient of 0 to 15% methanol in dichloromethanewith 0.5% acetic acid to elute the desired product; MS (−): m/z 321.00(M−1)

Example D-30 [(4-Hydroxy-7-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was prepared from[(1-Chloro-4-hydroxy-7-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid, Example D-34, following a procedure analogous to that described indetail in Example D-37. The final product was purified by chromatographyon silica gel using a gradient of 0 to 15% methanol in dichloromethanewith 0.5% acetic acid to elute the desired product; MS (−): m/z 321.02(M−1)

Example D-31[(1-Chloro-4-hydroxy-6-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid a) (5-Bromo-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid methylester

50.3 g of 4-bromophthalimide, 92.0 g of potassium carbonate, and 24.5 mlof methyl bromoacetate were added to 888 ml of acetone. The resultantmixture was heated to reflux temperature for 24 h, and then cooled toroom temperature. The mixture was filtered through a fine glass frit toremove all solid material, and the solution was then concentrated undervacuum to provide 66 g of the desired product, a white solid; ¹H NMR(CDCl₃): δ=3.76 (s, 3H), 4.43 (s, 2H), 7.71-7.75 (m, 1H), 7.85-7.90 (dd,1H), 8.00 (m, 1H).

b) (1,3-Dioxo-5-phenyl-1,3-dihydro-isoindol-2-yl)-acetic acid methylester

6.0 g of the above bromo-phthalimide product was dissolved in 70 ml ofethylene glycol dimethyl ether. To the solution was added 3.7 g ofphenyl boronic acid, 13 g cesium carbonate, and 2 gtetakis(triphenylphosphine)palladium(0). The mixture was stirred under anitrogen atmosphere at 65° C. for 48 h. The resultant mixture was pouredinto 250 ml of half saturated aqueous sodium bicarbonate solution, andthen extracted with 200 ml portions of ethyl acetate three times. Thecombined organic fractions were successively washed with 200 ml ofwater, saturated sodium bicarbonate, and brine solutions, and then driedover sodium sulfate. The solution was concentrated to a residue (11 g),which was purified by chromatography on silica gel using a gradient of 0to 25% ethyl acetate in hexanes to elute the desired product. 1.1 g ofpurified product was obtained; MS (+): m/z 296.02 (M+1)

c) 1,4-Dihydroxy-7-phenyl-isoquinoline-3-carboxylic acid butyl ester (A)and 1,4-Dihydroxy-6-phenyl-isoquinoline-3-carboxylic acid butyl ester(B)

1.4 g of the above product was added to 18.8 ml solution of 0.5 N sodiumn-butoxide in n-butanol. The resultant mixture was heated to 100° C. for2 h, and then cooled to room temperature. The mixture was poured into a100 ml solution of 0.5 N aqueous hydrochloric acid solution, andextracted with 100 ml portions of ethyl acetate three times. Thecombined organic extracts were filtered to remove any insoluble materialand then washed successively with water and brine. The solution wasdried over sodium sulfate and concentrated under vacuum to a residue(1.1 g), which was purified by chromatography on silica gel using agradient of 0 to 20% ethyl acetate in dichloromethane to elute two majorproducts (R_(f) isomer A=0.64, R_(f) B=0.48; 15% Ethyl acetate: 85%Dichloromethane)

Isomer A: 397 mg; MS (+) m/z 388.11 (M+1)

Isomer B: 195 mg; MS (+) m/z 388.10 (M+1)

d) 1-Chloro-4-hydroxy-6-phenyl-isoquinoline-3-carboxylic acid butylester

The title compound was prepared using the above isomer B,1,4-Dihydroxy-6-phenyl-isoquinoline-3-carboxylic acid butyl ester, underconditions analogous to those described in detail in Example D-39.d; MS(+): m/z 356.06 (M+1)

e) [(1-Chloro-4-hydroxy-6-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was obtained as follows: 95 mg of the above ester and300 mg of glycine were suspended in a solution of 5.4 ml of 0.5 sodiummethoxide in methanol. The mixture was heated to reflux temperature for42 h, and then cooled to room temperature. The mixture was diluted with30 ml of aqueous bicarbonate and washed with 30 ml ethyl acetate. Theaqueous solution was acidified to pH 3 with 6 N aqueous hydrochloricacid, and then extracted with 35 ml ethyl acetate three times. Thecombined organic extracts were dried over sodium sulfate andconcentrated under vacuum to provide 73 mg of the desired product, awhite solid; MS (−): m/z 354.99 (M−1)

Example D-32[(1-Chloro-4-hydroxy-7-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was prepared from1,4-Dihydroxy-7-phenyl-isoquinoline-3-carboxylic acid butyl ester,Example D-33c isomer A, following procedures analogous to thosedescribed in detail in examples D-39d and D-39e;

1-Chloro-4-hydroxy-7-phenyl-isoquinoline-3-carboxylic acid butyl ester;MS (+): m/z 356.09 (M+1)

[(1-Chloro-4-hydroxy-7-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid; MS (−): m/z 355.01 (M−1)

Example D-33[(1-Bromo-4-hydroxy-6-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid

161 mg of 1,4-Dihydroxy-6-phenyl-isoquinoline-3-carboxylic acid butylester, Example D-33c isomer B, was suspended in 3 ml of anhydrousacetonitrile. 896 mg of phosphorous oxybromide was added, and themixture was heated to reflux temperature for 5 h. The mixture was cooledto room temperature, concentrated to a residue under reduced pressure,and suspended in a mixture of 40 ml ethyl acetate and 40 ml ofhalf-saturated aqueous sodium bicarbonate. The biphasic mixture wasrapidly stirred for 10 min., and then was extracted with 40 ml portionsof ethyl acetate three times. The combined organic extracts wereconcentrated under vacuum and purified by chromatography on silica gelusing a gradient of 0-5% ethyl acetate in dichloromethane to elute onemajor fraction. 26 mg of material was recovered and used directly in thenext reaction.

The residue and 58 mg of glycine were suspended in a solution of 1.4 mlof 0.5 sodium methoxide in methanol. The mixture was heated to refluxfor 18 h, then cooled to room temperature, and concentrated to ca. 0.5ml under reduced pressure. The mixture was diluted with 30 ml of waterand acidified to pH 3 with 6 N aqueous hydrochloric acid. The resultingprecipitate was collected and washed with cold water two times. Thesolid product was dried under vacuum to yield 16 mg of the desiredproduct; MS (−): m/z 398.90, 400.92 (M−1, M+1; Br isotopes)

Example D-34[(1-Bromo-4-hydroxy-7-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid

The title compound was prepared from1,4-Dihydroxy-7-phenyl-isoquinoline-3-carboxylic acid butyl ester,Example D-33c isomer A, using conditions analogous to those described indetail in Example D-35. The final product was purified by chromatographyon silica gel using a gradient of 0 to 10% methanol in dichloromethanewith 0.5% acetic acid to elute the desired product; MS (−): m/z 398.91,400.95 (M−1, M+1; Br isotopes)

Example D-35 [(4-Hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid

200 mg of[(1-Chloro-4-hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid, from Example D-39.e, was suspended in a solution of 12 ml MeOH and4 ml water. 45 mg of sodium carbonate and 100 mg of palladium 10 wt. %on activated carbon were added, and the mixture was stirred for 18 hunder a hydrogen atmosphere provided by a hydrogen filled balloon. Theresultant mixture was diluted with methanol and aqueous sodiumbicarbonate and then filtered through a celite pad. The solution wasconcentrated under reduced pressure to ca. 6 ml, then diluted to 30 mlwith half saturated bicarbonate solution, and then acidified to pH 3with concentrated aqueous hydrochloric acid. The aqueous solution wasextracted with 30 ml portions of ethyl acetate three times. The combinedorganic extracts were dried over sodium sulfate and concentrated underreduced pressure to provide 107 mg of the desired product as a whitesolid; MS (+): m/z 323.08 (M+1)

Example D-36 [(4-Hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was prepared from[(1-Chloro-4-hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid, Example D-40, using conditions analogous to those described indetail in Example D-37; MS (+): m/z 323.06 (M+1)

Example D-37[(1-Chloro-4-hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid a) Biphenyl-2,3-dicarboxylic acid

15 g of 2-methyl-3-biphenylmethanol and 75 mg cetyltrimethylammoniumbromide were added to 150 ml of water, and the resultant mixture wascooled to 0° C. in an ice bath. 48 g of potassium permanganate was addedto the cold mixture and reaction was stirred at 0° C. for 10 min., atroom temperature for 16 h, then at 70° C. for 48 h. The clear solutioncontaining black solid was filtered through a pad of celite, and washedwith 100 ml of dichloromethane. The aqueous solution was then acidifiedto pH 3 with 6 N aqueous hydrochloric acid and extracted four times with150 ml portions of ethyl acetate. The combined organic fractions weredried over anhydrous sodium sulfate, and concentrated to 12.9 g ofproduct; ¹H NMR (d6-DMSO): δ=7.28-7.46 (m, 5H), 7.51-7.61 (m, 2H),7.84-7.89 (dd, 1H), 13.0 (s, 2H).

b) (1,3-Dioxo-4-phenyl-1,3-dihydro-isoindol-2-yl)-acetic acid methylester

10.5 g of the above di-acid and 3.25 g of glycine were mixed with amortar and pestle and then heated in an oil bath kept between 210 to230° C. for 15 min. The mixture was cooled and the resultant solid wasused directly in the next reaction.

To a solution of the crude phthalimide product, from the above reaction,in 125 ml of acetone was added 7.4 g of potassium carbonate and 5.7 mlof methyl sulfate. The mixture was heated to reflux for 24 h, and thencooled to room temperature. The mixture was diluted with 500 ml of waterand extracted with 500 ml of ethyl acetate three times. The combinedorganic fractions were washed with brine and dried over sodium sulfate.The solution was concentrated and resultant solid was crystallized fromethyl acetate to yield 5.0 g of a pale yellow solid; ¹H NMR (CDCl₃):δ=3.74 (s, 3H), 4.40 (s, 2H), 7.91-7.43 (m, 8H)

c) 1,4-Dihydroxy-8-phenyl-isoquinoline-3-carboxylic acid butyl ester (A)and 1,4-Dihydroxy-5-phenyl-isoquinoline-3-carboxylic acid butyl ester(B)

5.07 g of the above product was added to 68.8 ml of 0.5 N sodiumn-butoxide in n-butanol. The resultant mixture was heated to 95° C. for4 h, and then cooled to room temperature. 2.1 ml of acetic acid wasadded and the mixture was concentrated under reduced pressure to ca. 15ml volume. The crude products were diluted with half-saturated sodiumbicarbonate solution and extracted with ethyl acetate three times. Thecombined organic fractions were washed with water, then brine, and weredried over sodium sulfate. The solution was concentrated and the residue(5.3 g) was purified by chromatography on silica gel using a gradient of0 to 25% ethyl acetate in dichloromethane to elute two major fractions(R_(f) isomer A=0.68, R_(f) B=0.52, 15% ethyl acetate: 85%dichloromethane):

Isomer A, 2.19 g; MS (+) m/z 338.15 (M+1)

Isomer B, 1.22 g; MS (+) m/z 388.04 (M+1)

d) 1-Chloro-4-hydroxy-5-phenyl-isoquinoline-3-carboxylic acid butylester

500 mg of isomer B from the above reaction was suspended in 5 ml ofphosphorous oxychloride and heated to 100° C. for 1 h. The reactionmixture was cooled, concentrated to a residue under reduced pressure,and then diluted with 30 ml of water and 30 ml of ethyl acetate whilerapidly stirring. The pH of the aqueous phase was monitored and adjustedto ca. pH 7 with the addition of sodium bicarbonate. The biphasicmixture was stirred for 30 min. and then extracted with 30 ml of ethylacetate 3 times. The combined organic fractions were washed withsaturated sodium bicarbonate solution and brine, and then dried oversodium sulfate. The solution was concentrated under reduced pressure,and the residue (494 mg) was purified was purified by chromatography onsilica gel using a gradient of 5 to 20% ethyl acetate in dichloromethaneto elute one major fraction. 442 mg of product was obtained; MS: (+) m/z355.99 (M+1)

e) [(1-Chloro-4-hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid

435 mg of the above ester and 1.0 g of glycine were suspended in asolution of 24.4 ml of 0.5 N sodium methoxide in methanol. The mixturewas heated to reflux for 18 h, then cooled to room temperature, andconcentrated to ca. 5 ml under reduced pressure. The mixture was dilutedwith 50 ml of water and acidified to pH 3 with 1 N aqueous hydrochloricacid. The resulting precipitate was collected and washed with cold watertwo times. The solid product was dried under vacuum to yield 414 mg ofproduct; MS (+) m/z 356.99 (M+1)

Example D-38[(1-Chloro-4-hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was prepared from1,4-Dihydroxy-8-phenyl-isoquinoline-3-carboxylic acid butyl ester,Example D-39c isomer A, using conditions analogous to those described indetail in examples D-39d and D-39e.

1-Chloro-4-hydroxy-8-phenyl-isoquinoline-3-carboxylic acid butyl ester;MS (+): m/z 356.05 (M+1)

[(1-Chloro-4-hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid; MS (+): m/z 356.99 (M+1)

Example D-39[(1-Bromo-4-hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acida) 1-Bromo-4-hydroxy-5-phenyl-isoquinoline-3-carboxylic acid butyl ester

411 mg of 1,4-Dihydroxy-5-phenyl-isoquinoline-3-carboxylic acid butylester, from Example D-39c isomer B, was suspended in 15 ml of anhydrousacetonitrile. 2.0 g of phosphorous oxybromide was added and the reactionmixture was heated to reflux for 3.5 h. The reaction mixture was cooledand poured into 75 ml of 0° C. saturated aqueous sodium bicarbonatesolution. The mixture was stirred for 5 min and then extracted with 75ml portions of ethyl acetate three times. The combined organic fractionswere washed with brine, dried over sodium sulfate, and concentratedunder reduced pressure. The residue (434 mg) was purified bychromatography on silica gel using a gradient of 0 to 25% ethyl acetatein hexanes to elute the product as one major fraction. 480 mg of thedesired product was obtained; MS (+): m/z 422.02 (M+23)

b) [(1-Bromo-4-hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was obtained as follows: 200 mg of the above esterand 412 mg of glycine were suspended in a solution of 10 ml of 0.5 Nsodium methoxide in methanol. The mixture was heated to reflux for 24 h,then cooled to room temperature, and concentrated to ca. 3 ml underreduced pressure. The mixture was diluted with 50 ml of water andacidified to pH 3 with 1 N aqueous hydrochloric acid. The resultingprecipitate was collected and washed with cold water two times. Thesolid product was dried under vacuum to yield 188 mg of product; MS (−):m/z 398.96, 400.95 (M−1, M+1; Br isotopes)

Example D-40[(1-Bromo-4-hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid

The title compound was prepared from1,4-Dihydroxy-8-phenyl-isoquinoline-3-carboxylic acid butyl ester,Example D-39c isomer A, using conditions analogous to those described indetail in Example D-41;

1-Bromo-4-hydroxy-8-phenyl-isoquinoline-3-carboxylic acid butyl ester;MS (+): m/z 400.00, 402.03 (M+1, M+3; Br isotopes)

[(1-Bromo-4-hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid; MS (−): m/z 398.95, 400.98 (M−1, M+1; Br isotopes)

Example D-41[(1-Ethylsulfanyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acida) 1-Ethylsulfanyl-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

52 mg of 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester,Example D-20c, was dissolved in 2 ml of ethanethiol and heated in asealed tube at 70° C. for 24 h, and 100° C. for 48 h. The resultantsolution was concentrated under vacuum and the residue (54 mg) waspurified by chromatography on silica gel using a gradient of 0 to 20percent ethyl acetate in hexanes to elute the product. 25 mg of productwas obtained; MS (+): m/z 306.06 (M+1)

b) [(1-Ethylsulfanyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was obtained using the ester above, under conditionsanalogous to those described in detail in Example D-39e; MS (−) m/z304.98 (M−1)

Example D-42{[4-Hydroxy-1-(4-methoxy-phenylsulfanyl)-isoquinoline-3-carbonyl]-amino}-aceticacid

To a solution of 100 mg of[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid, (U.S.Pat. No. 6,093,730, disclosed asN-((1-Chloro-4-hydroxyisoquinoline-3-yl)carbonyl)glycine), in 1 mlN,N-dimethylformamide was added 1 ml of 4-methoxybenzenethiol. Thesolution was heated at 120 to 130° C. in a sealed tube for 72 h. Thesolution was then concentrated under vacuum. The resultant residue (76mg) was purified by chromatography on silica gel using a gradient of 0to 15% methanol in dichloromethane with 0.5% acetic acid to elute theproduct. 6 mg of product was obtained; MS (+) m/z 385.05 (M+1)

Example D-43[(1-Chloro-4-hydroxy-7-iodo-isoquinoline-3-carbonyl)-amino]-acetic acida) (5-Iodo-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid

4-Iodo-phthalic acid, 10 g, was mixed intimately with 2.63 g of glycineand the mixture was heated to 200° C. for 10 min. After cooling, thesolid reaction mixture was extracted with ethyl acetate to give, afterconcentration, 6.40 g of tan solid: MS-(−)-ion, Proton NMR (200 MHz,methanol-d-4): δ 8.26-8.18 (m, 2H), 7.68-7.61 (m, 1H), 4.39 (s, 1H).

b) (5-Iodo-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid methyl ester

6.4 g of the carboxylic acid product of Example D-55 a) were esterifiedfor 3 h with 2.7 g of dimethyl sulfate and 3.0 g of potassium carbonatein 25 ml of refluxing acetone. The reaction mixture was diluted withethyl acetate, filtered, and concentrated. The residue was dissolved infresh ethyl acetate and the organic layer was washed (water, brine), anddried over sodium sulfate. Concentration of the dry filtered ethylacetate solution gave 5.8 g of a light yellow solid: Proton NMR (200MHz, chloroform-d): δ 8.24-8.20 (m, 1H), 8.14-8.06 (m, 1H), 7.62-7.56(d, 1H), 4.40 (s, 2H), 3.75 (s, 3H).

c) 4-Hydroxy-7-iodo-1-oxo-1,2-dihydroisoquinoline-3-carboxylic acidbutyl ester and4-hydroxy-6-iodo-1-oxo-1,2-dihydro-isoquinoline-3-carboxylic acid butylester

Freshly cut sodium metal, 0.40 g, was dissolved in 22 ml of n-butanol at65° C. under a nitrogen atmosphere. A mixture of 3.0 g of(5-iodo-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid methyl ester in22 ml of n-butanol was added to the sodium butoxide solution and thereaction mixture was heated to 80° C. for 2 h. The cooled reactionmixture was acidified with 100 ml of 1 M hydrochloric acid to give asolid precipitate. The solid was collected by filtration and separatedby silica gel chromatography (eluant; 19:1 dichloromethane:ethylacetate) to give 0.219 g of4-hydroxy-7-iodo-1-oxo-1,2-dihydro-isoquinoline-3-carboxylic acid butylester: MS-(−)-ion, M−1=386.0 amu, and 0.150 g of4-hydroxy-6-iodo-1-oxo-1,2-dihydro-isoquinoline-3-carboxylic acid butylester: MS-(−) ion, M−1=386.0 amu.

d) 1-Chloro-4-hydroxy-7-iodo-isoquinoline-3-carboxylic acid butyl ester

0.215 g of 4-hydroxy-7-iodo-1-oxo-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester were added to 5 ml of POCl₃ at room temperature. Themixture was refluxed for 3 h and POCl₃ was removed under vacuum. Theresidue was dissolved in ethyl acetate and the solution was washed withsatd. aqueous sodium bicarbonate, dried (MgSO₄), filtered, andconcentrated to give 0.205 g of a while solid: Proton NMR (200 MHz,chloroform-d) δ 11.91 (s, 1H), 8.67 (m, 1H), 8.10 (m, 2H), 4.49 (t, J=7Hz, 2H), 1.95-1.75 (m, 2H), 1.60-1.39 (m, 2H), 1.00 (t, J=7 Hz, 3H).

e) [(1-Chloro-4-hydroxy-7-iodo-isoquinoline-3-carbonyl)-amino]-aceticacid

0.095 g of 1-chloro-4-hydroxy-7-iodo-isoquinoline-3-carboxyline acidbutyl ester were added to a mixture of 0.263 g of glycine in 4.7 ml of0.5M sodium methoxide and the reaction mixture was refluxed for 18 h.The mixture was concentrated, the residue was dissolved in water, andthe solution was acidified with 1M hydrochloric acid. The precipitatewas extracted with ethyl acetate and the organic layer was washed withwater, dried (MgSO₄), filtered, and concentrated to give 0.079 g of apale yellow product MS-(−)-ion, M−1=406.9 amu.

Example D-44[(1-Chloro-4-hydroxy-6-iodo-isoquinoline-3-carbonyl)-amino]-acetic acida) 1-Chloro-4-hydroxy-6-iodo-isoquinoline-3-carboxylic acid butyl ester

Analogously to Example D-43 d), 0.150 g of4-hydroxy-6-iodo-1-oxo-1,2-dihydro-isoquinoline-3-carboxylic acid butylester was allowed to react with 5 ml of POCl₃ to afford 0.057 g of apale white solid: Proton NMR (200 MHz, chloroform-d): δ 11.9 (s, 1H),8.89 (m, 1H), 8.1 (m, 1H), 7.97 (m, 1H), 4.5 (t, J=7 Hz, 2H), 2.0-1.8(m, 2H), 1.65-1.4 (m, 2H), 1.00 (t, J=7 Hz, 3H).

b) [(1-Chloro-4-hydroxy-6-iodo-isoquinoline-3-carbonyl)-amino]-aceticacid

0.053 of the butyl ester from Example D-44 a) were allowed to react witha mixture of 0.147 g of glycine in 2.6 ml of a 0.5M solution of sodiummethoxide in methanol under conditions analogous to Example D-55 e) togive 0.047 g of product as an off-white solid: MS-(−)-ion, M−1=406.9amu.

Example D-45 [(4-Hydroxy-7-iodo-isoquinoline-3-carbonyl)-amino]aceticacid a) 4-Hydroxy-7-iodo-isoquinoline-3-carboxylic acid butyl ester

0.100 G of the product from Example D-43 d) were dissolved in 1.5 ml ofglacial acetic acid containing 0.015 g of red phosphorous and 56microliters of hydroiodic acid (d=1.701 g/ml). The reaction mixture wasrefluxed for 1 h, diluted with ethyl acetate, and filtered through aCelite plug. The filtrate was washed with satd. aqueous sodiumthiosulfate and satd. aqueous sodium bicarbonate, dried (MgSO₄),filtered, and concentrated to give a crude product. Silica gelchromatography of the crude product (eluant, 99:1 CH₂Cl₂-ethyl acetate)gave 0.073 g of a white solid: Proton NMR (200 MHz, chloroform-d): δ11.9 (s, 1H), 8.70 (s, 1H), 8.40-8.30 (m, 1H), 8.12-8.05 (m, 1H),8.05-7.96 (m, 1H), 4.48 (t, J=7 Hz, 2H), 1.95-1.80 (m, 2H), 1.60-1.40(m, 2H), 0.99 (t, J=7 Hz, 3H).

b) [(4-Hydroxy-7-iodo-isoquinoline-3-carbonyl)-amino]-acetic acid

0.042 g was obtained by allowing the butyl ester from Example D-45 a) toreact with a mixture of 0.142 g of glycine in 2.5 ml of 0.5M methanolicsodium methoxide analogously to Example D-55 e): MS-(−)-ion, M−1=3.73.0amu.

Example D-46[(1-Bromo-4-hydroxy-7-methyl-isoquinoline-3-carbonyl)-amino]-acetic acida) 4-Hydroxy-7-methyl-1-oxo-1,2-dihydro-isoquinoline-3-carboxylic acidbutyl ester

5.0 g of 4-methyl-phthalic acid gave, after a sequence of reactionanalogous to Examples D-43 a)-D-43 c), 0.213 g of4-hydroxy-7-methyl-1-oxo-1,2-dihydro-isoquinoline-3-carboxylic acidbutyl ester: MS-(−)-ion, M−1=274.1 amu.

b) 1-Bromo-4-hydroxy-7-methyl-isoquinoline-3-carboxylic acid butyl ester

0.210 g of the ester product from Example D-46 a) was added to 3.5 ml ofacetonitrile. Phosphorus oxybromide, 1.52 g, was added and the mixturewas refluxed for 6 h, cooled, and dissolved in ethyl acetate. The ethylacetate solution was washed with satd. aqueous NaHCO₃, dried (MgSO₄),filtered, and concentrated to give 0.266 g of a crude product. Silicagel chromatography of the crude material (eluant: methylene chloride)gave 0.094 g of a white solid: Proton NMR (200 MHz, chloroform-d): δ11.85 (s, 1H), 8.30-8.20 (d, 1H), 8.00 (br s, 1H), 7.70-7.60 (m, 1H),4.47 (t, J=7 Hz, 2H), 2.62 (s, 3H), 1.95-1.75 (m, 2H), 1.60-1.35 (m,2H), 1.00 (t, J=7 Hz, 3H).

c) [(1-Bromo-4-hydroxy-7-methyl-isoquinoline-3-carbonyl)-amino]-aceticacid

0.094 g of butyl ester from Example D-46 b) were allowed to react with amixture of 0.312 g of glycine in 5.5 ml of 0.5M methanolic sodiummethoxide analogously to Example D-55 e) to give 0.083 g of an off-whitesolid: MS-(−)-ion, M−1=339.0 amu.

Example D-47[(1-Bromo-7-butoxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acida) 1-Bromo-7-butoxy-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

0.150 g of7-butoxy-4-hydroxy-1-oxo-1,2-dihydro-isoquinoline-3-carboxylic acidbutyl ester, were allowed to react with phosphorous oxybromideanalogously to Example D-46 b) to give 0.105 g of an off-white solid:Proton NMR (200 MHz, chloroform-d): δ 11.82 (s, 1H), 8.68 (s, 1H), 8.26(d, 1H), 7.35 (dd, 1H), 7.19 (d, 1H), 4.49 (t, J=7 Hz, 2H), 4.12 (t, J=7Hz, 2H), 1.95-1.75 (m, 4H), 1.70-1.40 (m, 4H), 1.05-0.95 (m, 6H).

b) [(1-Bromo-7-butoxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

0.100 g of butyl ester from Example D-47 a) were allowed to react with amixture of glycine in methanolic sodium methoxide analogously to ExampleD-11 e) to give 0.094 g of a white solid: MS-(−)-ion, M−1=397.0 amu.

Example D-48[(1-Bromo-6-butoxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acida) 1-Bromo-6-butoxy-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

0.175 g of 6-butoxy-4-hydroxy-isoquinoline-3-carboxylic acid butylester, ¹ were allowed to react with phosphorous oxybromide analogouslyto Example D-46 b) to give 0.073 g of a white solid: Proton NMR (200MHZ, chloroform-d): δ 11.84 (s, 1H), 8.13 (d, 1H), 7.60 (m, 1H),7.42-7.35 (m, 1H), 4.48 (t, J=7 Hz, 2H), 4.15 (t, J=7 Hz, 2H), 1.95-1.75(m, 4H), 1.65-1.40 (m, 4H), 1.05-0.95 (m, 6H).

b) [(1-Bromo-6-butoxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

0.068 g of butyl ester from Example D-48 a) were allowed to react withglycine in methanolic sodium methoxide analogously to Example D-43 e) togive 0.063 g of an off-white solid: MS-(−)-ion, M−1=397.0 amu.

Example D-49[(6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-methyl-amino]-aceticacid

6-Benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid, 0.33 g,0.5 ml of triethylamine, 0.400 g of HATU, and 0.165 g of ethylN-methyl-amino-acetate hydrochloride were combined in 15 ml ofdichloromethane and the reaction mixture was stirred at room temperaturefor 18 h to give, after silica gel chromatography, 0.232 g of anoff-white solid, MS-(+)-ion: 429.0 amu. 0.208 g of this intermediateproduct were dissolved in 10 ml of methanolic NaOH (1.5 M) and themixture was stirred at room temperature for 3 h. The solvent was removedwith a rotary evaporator, the residue was dissolved in water, and theaqueous layer was extracted with 50 ml of ethyl acetate. The aqueouslayer was acidified to pH=1 with aqueous HCl to give a solidprecipitate. The solid was collected by suction filtration, washed withwater, and dried in a vacuum oven (80° C.) to give 0.180 g of whitesolid: MS-(+)-ion: 401.0 amu.

Example D-50[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-methyl-amino]-acetic acid

Prepared from 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acidanalogously to Example D-49: MS-(+)-ion: 294.9 amu.

Example D-51[(1-Chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-methyl-amino]-aceticacid

Prepared from 1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylicacid analogously to Example D-49: MS-(+)-ion: 353.0 amu.

Example D-52[(1-Chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-methyl-amino]-aceticacid

Prepared from 1-chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carboxylicacid analogously to Example D-49: MS-(+)-ion: 353.0 amu.

Example D-53[Carboxymethyl-(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared from 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid and(ethoxycarbonylmethyl-amino)-acetic acid ethyl ester analogously toExample D-49: MS-(+)-ion: 339.0 amu.

Example D-54[Carboxymethyl-(1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared from 1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carboxylicacid and (ethoxycarbonylmethyl-amino)-acetic acid ethyl esteranalogously to Example D-49 MS-(+)-ion: 397.0 amu.

Example D-55{[4-Hydroxy-1-(naphthalen-2-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-Hydroxy-1-(naphthalen-2-yloxy)-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butylester and Naphthalen-2-ol in analogy to Example D-20 d); MS-(+)-ion:M+1=388.1.

b){[4-Hydroxy-1-(naphthalen-2-yloxy)-isoquinoline-3-carbonyl]amino}-aceticacid

Synthesized from4-hydroxy-1-(naphthalen-2-yloxy)-isoquinoline-3-carboxylic acid butylester in analogy to Example D-1 g); MS-(+)-ion: M+1=389.1.

Example D-56{[4-Hydroxy-1-(pyridin-3-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-Hydroxy-1-(pyridin-3-yloxy)-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butylester and pyridin-3-ol in analogy to Example D-20 d); MS-(+)-ion:M+1=339.1.

b) {[4-Hydroxy-1-(pyridin-3-yloxy)-isoquinoline-3-carbonyl]amino}-aceticacid

Synthesized from 4-hydroxy-1-(pyridin-3-yloxy)-isoquinoline-3-carboxylicacid butyl ester in analogy to Example D-1 g); MS-(+)-ion: M+1=340.1.

Example D-57{[4-Hydroxy-1-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-Hydroxy-1-(4-methoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butylester and 4-methoxy-phenol in analogy to Example D-20 d); MS-(+)-ion:M+1=368.1.

b){[4-Hydroxy-1-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

Synthesized from4-hydroxy-1-(4-methoxy-phenoxy)-isoquinoline-3-carboxylic acid butylester in analogy to Example D-1 g); MS-(+)-ion: M+1=369.1.

Example D-58{[4-hydroxy-1-(3-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-hydroxy-1-(3-methoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butylester and 3-methoxy phenol in analogy to Example D-20 d); MS-(+)-ion:M+1=368.1.

b){[4-hydroxy-1-(3-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

Synthesized from4-hydroxy-1-(3-methoxy-phenoxy)-isoquinoline-3-carboxylic acid butylester in analogy to Example D-1 g); MS-(−)-ion: M−1=367.0.

Example D-59{[1-(3-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a) 1-(3-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butylester and 3-Fluoro phenol in analogy to Example D-20 d); MS-(+)-ion:M+1=356.1.

b){[1-(3-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Synthesized from1-(3-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester in analogy to Example D-1 g); MS-(+)-ion: M+1=357.09.

Example D-60{[1-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]amino}-aceticacid a) 1-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butylester and 4-fluoro phenol in analogy to Example D-20 d); MS-(+)-ion:M+1=356.1.

b){[1-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Synthesized from1-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester in analogy to Example D-1 g); MS-(+)-ion: M+1=357.0.

Example D-61{[1-(2-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a) 1-(2-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from 1-Chloro-4-hydroxy-isoquinoline-3-carboxylic acid butylester and 2-fluorophenol in analogy to Example D-20 d); MS-(+)-ion:M+1=356.1.

b){[1-(2-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Synthesized from1-(2-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester in analogy to Example D-1 g); MS-(+)-ion: M+1=357.11.

Example D-62{[4-Hydroxy-1-(2-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-Hydroxy-1-(2-methoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butylester and 2-methoxy phenol in analogy to Example D-20 d); MS-(+)-ion:M+1=368.13.

b){[4-Hydroxy-1-(2-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

Synthesized from4-hydroxy-1-(2-methoxy-phenoxy)-isoquinoline-3-carboxylic acid butylester in analogy to Example D-1 g); MS-(+)-ion: M+1=369.09.

Example D-63{[1-(4-Acetylamino-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-Acetoxy-1-(4-acetylamino-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

4-hydroxy-1-phenoxy-isoquinoline-3-carboxylic acid butyl ester (261 mg,0.77 mmol; see Example D-20 d) was dissolved in conc. H₂SO₄ (4 ml) atambient temperature. The solution was cooled to 0° C. and KNO₃ (79 mg,0.77 mmol) was added slowly with stirring. The mixture was stirred at 0°C. for 2 h before it was poured into ice water (100 ml) with stirring.The mixture was extracted with EtOAc (3×30 ml). The combined organicphases were washed with aqueous NaHCO₃ solution and brine, dried, andconcentrated in vacuo. The residue was dissolved in a mixture of EtOAc(20 ml) and MeOH (10 ml). Sodium acetate (70 mg, 0.85 mmol) and Pd/C (75mg, 10 wt. % Pd) were added and the mixture was stirred under aH₂-atmosphere (1 atm) at ambient temperature for 24 h. The mixture wasthen filtered through a pad of celite. Celite and filter cake werewashed with hot MeOH (3×4 ml) and the combined organic phases wereconcentrated in vacuo. 150 mg of the resulting residue (total: 380 mg)were dissolved in EtOAc (8 ml). Triethylamine (325 μl, 2.3 mmol) wasadded and the solution was cooled to 0° C. Then acetic anhydride (110μl, 1.15 mmol) was added slowly with vigorous stirring. The mixture wasallowed to warm up to ambient temperature over night and was thenstirred for another 20 h at ambient temperature. Subsequently, EtOAc (50ml) was added. The mixture was washed with aqueous NaHCO₃ solution andbrine, dried, and concentrated in vacuo. The residue was purified byflash column chromatography on silica gel using CH₂Cl₂: MeOH=100:1 to100:3 as the eluent to give 150 mg of4-Acetoxy-1-(4-acetylamino-phenoxy)-isoquinoline-3-carboxylic acid butylester; MS-(+)-ion: M+1=437.11.

b){[1-(4-Acetylamino-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

A mixture of4-Acetoxy-1-(4-acetylamino-phenoxy)-isoquinoline-3-carboxylic acid butylester (150 mg, 0.34 mmol), glycine (290 mg, 3.4 mmol), and 7.8 ml of a0.5 N solution of sodium methoxide in methanol (3.9 mmol) was refluxedover the weekend with stirring. Then the solvent was evaporated in vacuoand the residue dissolved in 25 ml of water. The pH of the solution wassubsequently adjusted to about 2 and the resulting slurry was extractedwith ethyl acetate (3×30 ml). The combined extracts were dried overMgSO₄ and evaporated in vacuo. Recrystallization of the residue frommethanol/CH₂Cl₂ gave 86 mg of the title compound; MS-(+)-ion:M+1=396.15.

Example D-64{[4-Hydroxy-1-(4-methanesulfonylamino-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

4-Hydroxy-1-phenoxy-isoquinoline-3-carboxylic acid butyl ester (261 mg,0.77 mmol; see Example D-20 d) was dissolved in conc. H₂SO₄ (4 ml) atambient temperature. The solution was cooled to 0° C. and KNO₃ (79 mg,0.77 mmol) was added slowly with stirring. The mixture was stirred at 0°C. for 2 h before it was poured into ice water (100 ml) with stirring.The mixture was extracted with EtOAc (3×30 ml). The combined organicphases were washed with aqueous NaHCO₃ solution and brine, dried, andconcentrated in vacuo. The residue was dissolved in a mixture of EtOAc(20 ml) and MeOH (10 ml). Sodium acetate (70 mg, 0.85 mmol) and Pd/C (75mg, 10 wt. % Pd) were added and the mixture was stirred under aH₂-atmosphere (1 atm) at ambient temperature for 24 h. The mixture wasthen filtered through a pad of celite. Celite and filter cake werewashed with hot MeOH (3×4 ml) and the combined organic phases wereconcentrated in vacuo. 150 mg of the resulting residue (total: 380 mg)were dissolved in CH₂Cl₂ (8 ml). Triethylamine (165 μl) was added andthe solution was cooled to −20° C. Then MeSO₂Cl (36 μl) was added slowlywith vigorous stirring. The mixture was allowed to warm up to ambienttemperature over night and was then stirred for another 20 h at ambienttemperature. Subsequently, EtOAc (50 ml) was added. The mixture waswashed with aqueous NaHCO₃ solution and brine, dried, and concentratedin vacuo. The residue was purified by flash column chromatography onsilica gel using CH₂Cl₂: MeOH=100:0 to 100:3 as the eluent. To thepurified product (168 mg) was added glycine (293 mg, 3.4 mmol), and 7.8ml of a 0.5 N solution of sodium methoxide in methanol (3.9 mmol) andthe mixture was refluxed over the weekend with stirring. Then thesolvent was evaporated in vacuo and the residue dissolved in 30 ml ofwater. The pH of the solution was subsequently adjusted to about 2 andthe resulting mixture was extracted with ethyl acetate (3×30 ml). Thecombined extracts were dried over MgSO₄ and evaporated in vacuo.Recrystallization of the residue from methanol/CH₂Cl₂ gave 89 mg of thetitle compound; MS-(+)-ion: M+1=432.12.

Example D-65[(4-Hydroxy-1-phenylamino-isoquinoline-3-carbonyl)-amino]-acetic acid a)4-Hydroxy-1-phenylamino-isoquinoline-3-carboxylic acid butyl ester

A mixture of 1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid butylester (810 mg, 2.5 mmol, Example D-28 a) and aniline (3 ml) was stirredin a pressure tube in a microwave oven at 150° C. for 20 min. Thereaction was repeated on the same scale. Both reaction mixtures werecombined, EtOAc (100 ml) was added and the mixture was washed with H₂O(5×30 ml, pH=1-2). The organic phase was dried and concentrated invacuo. The residue was purified by flash column chromatography on silicagel using hexanes/EtOAc as the eluent to give 770 mg of4-hydroxy-1-phenylamino-isoquinoline-3-carboxylic acid butyl ester;MS-(+)-ion: M+1=337.21.

b) [(4-Hydroxy-1-phenylamino-isoquinoline-3-carbonyl)-amino]-acetic acid

Synthesized from hydroxy-1-phenylamino-isoquinoline-3-carboxylic acidbutyl ester in analogy to Example D-1 g); MS-(+)-ion: M+1=338.14.

Example D-66{[4-Hydroxy-6-(pyridin-3-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-(Pyridin-3-yloxy)-phthalonitrile

A mixture of 4-nitro-phthalonitrile (3.46 g, 20 mmol), pyridin-3-ol(1.90 g, 20 mmol), K₂CO₃ (8.29 g, 60 mmol), and DMF (50 ml) was stirredat ambient temperature overnight. The reaction mixture was then combinedwith another batch of the same reaction performed on the same scale.Subsequently, the solid components were removed by filtration and thefiltrate was concentrated in vacuo. To the residue was added water andthe mixture was extracted with EtOAc. The organic phase was then washedwith brine, dried, and evaporated in vacuo. The residue wasrecrystallized from EtOAc/MeOH to give 8.3 g of the title compound; ¹HNMR (CDCl₃): δ=8.56 to 8.59 (m, 1H), 8.45 to 8.47 (m, 1H), 7.76 (d, 1H),7.42 to 7.44 (m, 2H), 7.22 to 7.32 (m, 2H).

b) 4-(Pyridin-3-yloxy)-phthalic acid

Synthesized from 4-(pyridin-3-yloxy)-phthalonitrile in analogy toExample D-1 a); MS-(+)-ion: M+1=260.2.

c) [1,3-Dioxo-5-(pyridin-3-yloxy)-1,3-dihydro-isoindol-2-yl]-acetic acid

Synthesized from 4-(pyridin-3-yloxy)-phthalic acid in analogy to ExampleD-1 b); MS-(+)-ion: M+1=299.25.

d) [1,3-Dioxo-5-(pyridin-3-yloxy)-1,3-dihydro-isoindol-2-yl]-acetic acidmethyl ester

Synthesized from[1,3-dioxo-5-(pyridin-3-yloxy)-1,3-dihydro-isoindol-2-yl]-acetic acid inanalogy to Example D-1 c); MS-(+)-ion: M+1=313.21.

e) 1,4-Dihydroxy-6-(pyridin-3-yloxy)-isoquinoline-3-carboxylic acidbutyl ester and1,4-Dihydroxy-7-(pyridin-3-yloxy)-isoquinoline-3-carboxylic acid butylester

Synthesized from[1,3-dioxo-5-(pyridin-3-yloxy)-1,3-dihydro-isoindol-2-yl]-acetic acidmethyl ester in analogy to Example D-1 d). However, after addition of 2NHCl (pH was adjusted to 8-9) the mixture was extracted three times withEtOAc. The combined org. phases were dried and concentrated in vacuo.The residue was treated with MeOH and stored overnight in arefrigerator. The precipitate formed was filtered, washed with a smallamount of cold MeOH and dried in vacuo to give a regioisomeric mixtureof the title compounds as a white solid.1,4-dihydroxy-6-(pyridin-3-yloxy)-isoquinoline-3-carboxylic acid butylester and 1,4-Dihydroxy-7-(pyridin-3-yloxy)-isoquinoline-3-carboxylicacid butyl ester were not separated; MS-(+)-ion: M+1=355.09.

1-Chloro-4-hydroxy-6-(pyridin-3-yloxy)-isoquinoline-3-carboxylic acidbutyl ester and1-Chloro-4-hydroxy-7-(pyridin-3-yloxy)-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from[1,4-dihydroxy-6-(pyridin-3-yloxy)-isoquinoline-3-carboxylic acid butylester and 1,4-dihydroxy-7-(pyridin-3-yloxy)-isoquinoline-3-carboxylicacid butyl ester regioisomeric mixture in analogy to Example D-43 d).The regioisomers were not separated; MS-(+)-ion: M+1=373.01.

g) 4-Hydroxy-6-(pyridin-3-yloxy)-isoquinoline-3-carboxylic acid butylester (A) and 4-Hydroxy-7-(pyridin-3-yloxy)-isoquinoline-3-carboxylicacid butyl ester (B)

Synthesized from1-chloro-4-hydroxy-6-(pyridin-3-yloxy)-isoquinoline-3-carboxylic acidbutyl ester and1-Chloro-4-hydroxy-7-(pyridin-3-yloxy)-isoquinoline-3-carboxylic acidbutyl ester regioisomeric mixture in analogy to Example D-7 f). Theregioisomers were separated by flash column chromatography on silica geleluting with CH₂Cl₂: EtOAc (90:10 to 80:20). Evaporation of the firstfraction yielded B; MS-(+)-ion: M+1=339.09. Evaporation of the secondfraction yielded A; MS-(+)-ion: M+1=339.10.

h){[4-Hydroxy-6-(pyridin-3-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

Synthesized from 4-hydroxy-6-(pyridin-3-yloxy)-isoquinoline-3-carboxylicacid butyl ester in analogy to Example D-1 g); MS-(+)-ion: M+1=340.06.

Example D-67{[4-Hydroxy-7-(pyridin-3-yloxy)-isoquinoline-3-carbonyl]amino}-aceticacid

Synthesized from 4-hydroxy-7-(pyridin-3-yloxy)-isoquinoline-3-carboxylicacid butyl ester (Example D-66 g) in analogy to Example D-1 g);MS-(+)-ion: M+1=340.06.

Example D-68 [(1-Chloro-4-methoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) [(1-Chloro-4-methoxy-isoquinoline-3-carbonyl)-amino]-acetic acidmethyl ester

A mixture of [(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid (56 mg, 0.2 mmol; can be obtained according to U.S. Pat. No.6,093,730, October 1998, Weidmann et al.), Me₂SO₄ (57 μl, 0.6 mmol),KHCO₃ (306 mg, 3 mmol) and acetone (4 ml) was refluxed with stirring for48 h. The solvent was evaporated after that time and water (4 ml) wasadded to the residue. The mixture was extracted with EtOAc (3×20 ml).The combined organic phases were dried over MgSO₄ and evaporated invacuo to give a brown oil. Purification by flash column chromatographyon silica gel using hexanes:EtOAc=7:3 as the eluent gave the titlecompound as a pale yellow oil (21 mg); MS-(+)-ion: M+1=308.9.

b) [(1-Chloro-4-methoxy-isoquinoline-3-carbonyl)-amino]-acetic acid

A mixture of [(1-chloro-4-methoxy-isoquinoline-3-carbonyl)-amino]-aceticacid methyl ester (21 mg, 0.07 mmol), KOH (23 mg, 0.35 mmol) and EtOH (1ml) was stirred at ambient temperature for 3 h. Then the solvent wasevaporated in vacuo. The residue was dissolved in water (2 ml) and thepH of the solution was adjusted to 2-3 by the addition of aqueous 1NHCl. The mixture was extracted with EtOAc (4×10 ml). The combined org.phases were dried over MgSO₄ and evaporated in vacuo to give the titlecompound as a slightly yellowish solid (18 mg); MS-(+)-ion: M+1=295.0.

Example D-69 [(1-Chloro-4-ethoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) [(1-Chloro-4-ethoxy-isoquinoline-3-carbonyl)-amino]-acetic acidethyl ester

A mixture of [(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid (56 mg, 0.2 mmol; can be obtained according to U.S. Pat. No.6,093,730, October 1998, Weidmann et al.), Et₂SO₄ (59 μl, 0.44 mmol),KHCO₃ (306 mg, 3 mmol) and Et₂CO (3 ml) was refluxed with stirring for18 h. Then the solvent was evaporated and water (4 ml) was added to theresidue. The mixture was stirred vigorously for 5 min before it wasfiltered. The filter cake was dissolved in EtOAc and the solution wasdried over MgSO₄. The solution was concentrated in vacuo. The resultingbrown solid was dissolved in EtOAc (0.5 ml) and hexanes was added. Themixture was stored for 14 h at ambient temperature before the solventwas decanted from precipitate formed. The precipitate was dried in vacuoto give the title compound as white crystals (8 mg); MS-(+)-ion:M+1=337.0.

b) [(1-Chloro-4-ethoxy-isoquinoline-3-carbonyl)-amino]-acetic acid

Synthesized from[(1-chloro-4-ethoxy-isoquinoline-3-carbonyl)-amino]-acetic acid ethylester in analogy to Example D-68 b); MS-(+)-ion: M+1=309.0.

Example D-70[(4-Hydroxy-1-methoxy-isoquinoline-3-carbonyl)-amino]-acetic acid a)4-Benzyloxy-1-hydroxy-isoquinoline-3-carboxylic acid butyl ester

A mixture of 1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester(26.13 g, 100 mmol; Example D-20 b), PhCH₂Br (18.2 ml, 150 mmol), MeONa(0.5 M in MeOH, 200 ml, 100 mmol) was stirred at ambient temperature for48 h. Then the solvent was evaporated and EtOAc (100 ml) was added tothe residue. The mixture was stirred vigorously for 10 min before it wasfiltered. The filtrate was washed with aqueous 2.5N NaOH (2×100 ml) andaqueous 2N HCl (1×100 ml). The organic phase was dried over MgSO₄ andevaporated in vacuo. The residue was recrystallized from MeOH (500ml)/water (300 ml). The resulting yellow solid was further purified byflash column chromatography on silica gel usinghexanes:EtOAc:NEt₃=65:30:5 as the eluent to give 10.8 g of a yellowsolid. 2 g of this material were further purified by flash columnchromatography on silica gel using hexanes:EtOAc:NEt₃=75:20:5 as theeluent to give 1.57 g of the title compound as a slightly yellowishsolid; ¹H NMR (CDCl₃): δ=8.88 (bs, 1H), 8.46 (d, 1H), 8.42 (d, 1H), 7.26to 7.96 (m, 7H), 5.06 (s, 2H), 4.38 (t, 2H), 1.69 (m, 2H), 1.37 (m, 2H),0.91 (t, 3H).

b) 4-Benzyloxy-1-methoxy-isoquinoline-3-carboxylic acid butyl ester

A mixture of 4-benzyloxy-1-hydroxy-isoquinoline-3-carboxylic acid butylester (1 eq.), Me₃OBF₄ (6 eq), KHCO₃ (14 eq) and CH₂Cl₂ (10 ml/mmol4-benzyloxy-1-hydroxy-isoquinoline-3-carboxylic acid butyl ester) wasstirred at ambient temperature for 24 h. Then water (10 ml/mmol) wasadded and the mixture was extracted with CH₂Cl₂ (40 ml/mmol). Theorganic phase was separated, dried over MgSO4 and evaporated in vacuo togive a yellowish solid. The crude product was purified by flash columnchromatography on silica gel using hexanes:EtOAc=85:15 as the eluent.Evaporation of the first fraction gave the title compound as a colorlessoil in 20% yield; ¹H NMR (CDCl₃): δ=8.21 to 8.25 (m, 1H), 8.05 to 8.09(m, 1 H), 7.33 to 7.73 (m, 7H), 5.13 (s, 2H), 4.38 (t, 2H), 4.16 (s,3H), 1.69 (m, 2H), 1.37 (m, 2H), 0.94 (t, 3H).

c) 4-Hydroxy-1-methoxy-isoquinoline-3-carboxylic acid butyl ester

A mixture of 4-benzyloxy-1-methoxy-isoquinoline-3-carboxylic acid butylester (164 mg, 0.45 mmol), Pd/C (50 mg, 10 wt % Pd) and EtOAc (15 ml)was stirred under a H₂-atmosphere at ambient pressure and temperaturefor 16 h. Then the mixture was filtered through a pad of celite. Celiteand filter cake were washed thoroughly with EtOAc and the combinedorganic phases were concentrated in vacuo to give the title compound asa white solid (115 mg); ¹H NMR (CDCl₃): δ=11.48 (s, 1H), 8.27 to 8.32(m, 1H), 8.17 to 8.21 (m, 1H), 7.65 to 7.78 (m, 2H), 4.43 (t, 2H), 4.10(s, 3H), 1.87 (m, 2H), 1.54 (m, 2H), 1.02 (t, 3H).

d) [(4-Hydroxy-1-methoxy-isoquinoline-3-carbonyl)-amino]-acetic acid

Synthesized from 4-hydroxy-1-methoxy-isoquinoline-3-carboxylic acidbutyl ester in analogy to Example D-1 g); MS-(−)-ion: M−1=275.0.

Example D-71 [(1-Ethoxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 1-Ethoxy-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

A mixture of 4-benzyloxy-1-hydroxy-isoquinoline-3-carboxylic acid butylester (422 mg, 1.2 mmol, Example D-70 a), KHCO3 (2.22 g, 22 mmol), and1M Et₃OBF₄ in CH₂Cl₂ (10 ml, 10 mmol) was stirred for 16 h at ambienttemperature and then was refluxed with stirring for another 3 days.According to TLC 4-Benzyloxy-1-hydroxy-isoquinoline-3-carboxylic acidbutyl ester did not react under these conditions. Therefore, additionalKHCO₃ (2.22 g, 22 mmol) and 1M Et₃OBF₄ in CH₂Cl₂ (10 ml, 10 mmol) wereadded and the mixture was concentrated in vacuo. Subsequently,1,2-dichloroethane (10 ml) was added and the mixture was refluxed withstirring for 16 h. Then the solvent was evaporated in vacuo. To theresidue was added water (25 ml) and the mixture was extracted with EtOAc(2×50 ml). The combined organic phases were dried over MgSO₄ andevaporated in vacuo to give a yellowish solid (374 mg). Purification byflash column chromatography on silica gel using hexanes:EtOAc=85:15 asthe eluent gave a yellowish oil (104 mg). The chromatographicalpurification was repeated using hexanes:EtOAc=99:2, and, subsequently99:1 as the eluent to give the title compound as a colorless oil (60mg); ¹H NMR (CDCl₃): δ=11.45 (s, 1H), 8.20 to 8.32 (m, 2H), 7.64 to 7.78(m, 2H), 4.38 to 4.59 (m, 4H), 1.84 (m, 2H), 1.54 (m, 5H), 1.01 (t, 3H).

b) [(1-Ethoxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid

Synthesized from 1-Ethoxy-4-hydroxy-isoquinoline-3-carboxylic acid butylester in analogy to Example D-1 g); MS-(+)-ion: M+1=291.0.

Example D-72 [(4-Acetoxy-1-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid a) [(1-Oxo-3-phenyl-1H-indene-2-carbonyl)-amino]-acetic acid methylester

A mixture of 1-oxo-3-phenyl-1H-indene-2-carboxylic acid (2.13 g, 8.5mmol; can be obtained according to M. R. Barvian et al. in Bioorg. Med.Chem. Lett. 1997, 7, 2903-2908) and SOCl₂ (17 ml) was refluxed withstirring for 15 min. Excess SOCl₂ was then evaporated in vacuo. Theresidue was dissolved in anhydrous CH₂Cl₂ (20 ml), and subsequently thesolution was concentrated in vacuo again to remove last traces of SOCl₂.The residue was dissolved in anhydrous CH₂Cl₂ (20 ml). The solution wascooled with an ice bath before glycine methyl ester hydrochloride (1.27g, 10 mmol) and subsequently NEt₃ (3.52 ml, 25 mmol, dropwise addition)were added with stirring. The ice bath was then removed and stirring wascontinued at ambient temperature for 45 min before the mixture wasconcentrated in vacuo. To the residue was added water (10 ml) andaqueous 2N HCl (15 ml) and the mixture was extracted with ethyl acetate(1×70 ml). The organic phase was dried over MgSO₄ and evaporated invacuo to give an orange solid (2.77 g). Purification by flash columnchromatography on silica gel using hexanes:EtOAc=2:1 as the eluent gavethe title compound as an orange solid (2.11 g); MS-(+)-ion: M+1=322.0.

b) [(4-acetoxy-1-phenyl-isoquinoline-3-carbonyl)-amino]acetic acidmethyl ester

[(1-Oxo-3-phenyl-1H-indene-2-carbonyl)-amino]-acetic acid methyl ester(1.864 g, 5.8 mmol) was dissolved in a mixture of concentrated H₂SO₄ (16ml) and glacial acetic acid (16 ml) at 50 to 60° C. Then NaN₃ (985 mg,15 mmol) was added in portions with stirring so that the temperature didnot exceed 60° C. Stirring was then continued at 50 to 60° C. foradditional 30 min before the mixture was poured onto ice (200 g). Theresulting mixture was basified by addition of concentrated aqueous NH₃(55 ml, D=0.89 g/ml) and extracted with CH₂Cl₂ (2×100 ml). The combinedorganic phases were dried over MgSO₄ and then filtered through silicagel. The filtrate was discarded. The silica gel was washed with EtOAc(ca. 400 ml). The resulting solution was concentrated in vacuo to give adark oil (250 mg). Further purification by flash column chromatographyon silica gel using EtOAc and then EtOAc:hexanes=7:3 as the eluent gavethe title compound as a tan solid (19 mg); ¹H NMR (CDCl₃): δ=7.11 to7.98 (m), 3.75 (d, 2H), 3.68 (s, 1H), 2.19 (s, 3H).

c) [(4-Acetoxy-1-phenyl-isoquinoline-3-carbonyl)-amino]acetic acid

A mixture of [(4-Acetoxy-1-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid methyl ester (3.8 mg, 0.01 mmol) and aqueous 6N HCl (1 ml) wasstirred at ambient temperature for 16 h before the pH of the solutionwas adjusted to ca. 8 by addition of concentrated aqueous NaHCO₃solution. The solution was washed with EtOAc (2×10 ml) before it wasacidified by addition of aqueous 2N HCl. Subsequently, the mixture wasextracted with EtOAc (2×10 ml). The combined organic phases were driedover MgSO₄ and evaporated in vacuo to give the title compound as ayellow oil (1.9 mg); MS-(+)-ion: M+1=364.9.

Example D-73 [(4-Hydroxy-1-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid a) 4-Hydroxy-1-phenyl-isoquinoline-3-carboxylic acid ethyl ester

A mixture of 4-acetoxy-1-phenyl-isoquinoline-3-carboxylic acid ethylester (671 mg, 2 mmol; can be obtained according to D. A. Walsh et al.in J. Med. Chem. 1978, 21, 582-585), n-BuOH (60 ml) and concentratedH₂SO₄ (1.7 ml) was refluxed with stirring for 4 h before the reactionmixture was added to concentrated aqueous NaHCO₃ solution (60 ml) withstirring. Then EtOAc (120 ml) was added and the mixture was stirredvigorously for 15 min. Subsequently, the organic phase was separated,dried over MgSO₄ and concentrated in vacuo. Purification of the residueby flash column chromatography on silica gel using hexanes:EtOAc=95:5 asthe eluent gave the title compound as a solid (126 mg); ¹H NMR (CDCl₃):δ=11.96 (s, 1H), 8.44 to 8.49 (m, 1H), 8.01 to 8.05 (m, 1H), 7.43 to7.80 (m, 7H), 4.56 (q, 2H), 1.49 (t, 3H).

b) [(4-Hydroxy-1-phenyl-isoquinoline-3-carbonyl)-amino]acetic acid

Synthesized from 4-Hydroxy-1-phenyl-isoquinoline-3-carboxylic acid ethylester in analogy to Example D-1 g); MS-(+)-ion: M+1=323.1.

Example D-74 [(1-Ethoxy-4-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid a) 1-Chloro-4-phenyl-isoquinoline-3-carboxylic acid ethyl ester

A mixture of 1-hydroxy-4-phenyl-isoquinoline-3-carboxylic acid ethylester (1.17 g, 4 mmol; can be obtained according to A. Marsili et al.,Ann. Chim. (Rome), 1962, 52, 112-120), and concentrated POCl₃ (10 ml)was refluxed with stirring for 1 h. Then the mixture was concentrated invacuo. The residue was dissolved in EtOAc (50 ml), concentrated aqueousNaHCO₃ solution (40 ml) was added and the mixture was stirred vigorouslyfor 1 h. Subsequently, the organic phase was separated, dried over MgSO₄and concentrated in vacuo to give the title compound as a yellowishsolid (1.20 g); MS-(+)-ion: M+1=312.0.

b) [(1-Chloro-4-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acidmethyl ester

A mixture of 1-chloro-4-phenyl-isoquinoline-3-carboxylic acid ethylester (1.184 g, 3.8 mmol) aqueous 2N NaOH (15 ml, 30 mmol) and EtOH (15ml) was refluxed with stirring for 2.5 h. Then the mixture wasconcentrated to ½ of its volume. Subsequently, the solution wasacidified by addition of concentrated HCl and the resulting suspensionwas extracted with EtOAc (2×50 ml). The combined organic phases weredried over MgSO₄ and evaporated in vacuo to give a yellowish solid(1.018 g). To 996 mg of this yellowish solid was added SOCl₂ (7 ml) andthe mixture was refluxed with stirring for 1 h. Excess SOCl₂ was thenevaporated in vacuo. The residue was dissolved in anhydrous CH₂Cl₂ (10ml), and subsequently the solution was concentrated in vacuo again toremove last traces of SOCl₂. The residue was dissolved in anhydrousCH₂Cl₂ (8 ml). The solution was cooled with an ice bath before glycinemethyl ester hydrochloride (507 mg, 4 mmol) and subsequently NEt₃ (1.55ml, 11 mmol, dropwise addition) were added with stirring. The ice bathwas then removed and stirring was continued at ambient temperature for 1h before the mixture was concentrated in vacuo. To the residue was addedwater (15 ml) and the mixture was extracted with ethyl acetate (1×50ml). The organic phase was dried over MgSO₄ and evaporated in vacuo togive a tan solid (1.07 g). Recrystallization from MeOH (30 ml)/water (10ml) gave the title compound as a slightly yellowish solid (430 mg);MS-(+)-ion: M+1=355.0.

c) [(1-Ethoxy-4-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid

A mixture of [(1-Chloro-4-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid methyl ester (177 mg, 0.5 mmol), KOH (325 mg, 5 mmol) and EtOH (10ml) was stirred at ambient temperature for 90 min before the solvent wasevaporated in vacuo. The residue was dissolved in water (10 ml). Thesolution was acidified by addition of concentrated aqueous HCl andextracted with EtOAc (2×15 ml). The combined organic phases were driedover MgSO₄ and concentrated in vacuo to give the title compound as aslightly yellowish solid (169 mg); MS-(+)-ion: M+1=351.0.

Example D-75 [(1-Chloro-4-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid

A mixture of [(1-chloro-4-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid methyl ester (50 mg, 0.14 mmol, Example D-74 b), and aqueous 6N HClwas stirred at ambient temperature for 11 days before the solution wasneutralized by the addition of concentrated aqueous NaHCO₃. The mixturewas extracted with EtOAc (50 ml). The organic phase was dried over MgSO₄and concentrated in vacuo to give the title compound as a white solid(35 mg); MS-(+)-ion: M+1=341.0.

Example D-76 [(4-Phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid a)[(4-Phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid methyl ester

A mixture of [(1-chloro-4-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid methyl ester (177 mg, 0.5 mmol, Example D-74 b), Pd/C (50 mg, 10 wt% Pd), sodium acetate (49 mg, 0.6 mmol), MeOH (10 ml) and EtOAc (5 ml)was stirred under a H₂-atmosphere at ambient pressure and temperaturefor 2 h. Then the mixture was filtered through a pad of celite. Celiteand filter cake were washed thoroughly with EtOAc and the combinedorganic phases were concentrated in vacuo. To the residue was addedconcentrated aqueous NaHCO₃ (10 ml) and the mixture was extracted withEtOAc (2×15 ml). The combined organic phases were dried over MgSO₄ andconcentrated in vacuo to give the title compound as a colorless gum (154mg); MS-(+)-ion: M+1=321.0.

b) [(4-Phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid

A mixture of [(4-Phenyl-isoquinoline-3-carbonyl)-amino]-acetic acidmethyl ester (144 mg, 0.45 mmol), KOH (325 mg, 5 mmol) and EtOH (10 ml)was stirred at ambient temperature for 18 h before the solvent wasevaporated in vacuo. The residue was dissolved in water. The pH of thesolution was adjusted to 3-4 by addition of concentrated aqueous HCl.The solution was then extracted with EtOAc (2×25 ml). The combinedorganic phases were dried over MgSO₄ and concentrated in vacuo to givethe title compound as a yellowish solid (127 mg); MS-(+)-ion: M+1=307.1.

Example D-77 [(4-Hydroxy-1-methyl-isoquinoline-3-carbonyl)-amino]-aceticacid a) 1-Bromo-4-hydroxy-isoquinoline-3-carboxylic acid

A mixture of 1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid butylester (8.18 g, 25 mmol; Example D-28 a) aqueous 2N NaOH (80 ml, 160mmol) and EtOH (80 ml) was refluxed with stirring for 2 h. Then thesolution was concentrated in vacuo to ½ of its volume, diluted withwater (200 ml), and was acidified by addition of concentrated aqueousHCl. After stirring at ambient temperature for 1 h the resultingsuspension was submitted to vacuum filtration. The filter cake waswashed thoroughly with water and dried in vacuo at 75° C. to give thetitle compound as a white solid (6.10 g); ¹H NMR (DMSO-d6): δ=8.30 to8.37 (m, 1H), 8.16 to 8.22 (m, 1H), 7.93 to 8.03 (m, 2H).

b) 4-Hydroxy-1-methyl-isoquinoline-3-carboxylic acid methyl ester

To a solution of 1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid (670mg, 2.5 mmol) in anhydrous THF (100 ml) was added slowly a 2.5 Msolution of n-BuLi in hexanes (4 ml, 10 mmol) at −78° C. with stirring.After stirring for another 5 min MeI (316 μl, 5 mmol) was added.Stirring was continued for additional 10 min at −78° C. before water (50ml) and aqueous 2N HCl (6 ml) were added. The mixture was allowed towarm up to ambient temperature with stirring and was then concentratedin vacuo to ca. ½ of its volume. The resulting precipitate was suckedoff, washed with water, dried in vacuo at 80° C. and was recrystallizedfrom EtOH to give a light tan solid (141 mg). A mixture of 102 mg of theaforementioned light tan solid, Me₂SO₄ (48 μl, 0.5 mmol), KHCO₃ (1.0 g,10 mmol) and acetone (10 ml) was refluxed with stirring for 15 h. Thenthe mixture was concentrated in vacuo. To the residue was added water(20 ml) and the mixture was extracted with EtOAc (3×20 ml). The combinedorganic phases were dried over MgSO₄ and concentrated in vacuo to givethe title compound as a tan solid; ¹H NMR (CDCl₃): δ=11.66 (s, 1H), 8.39to 8.44 (m, 1H), 8.02 to 8.09 (m, 1H), 7.74 to 7.81 (m, 2H), 4.08 (s,3H), 2.90 (s, 3H).

c) [(4-Hydroxy-1-methyl-isoquinoline-3-carbonyl)-amino]-acetic acid

Synthesized from 4-hydroxy-1-methyl-isoquinoline-3-carboxylic acidmethyl ester in analogy to Example D-1 g); MS-(−)-ion: M−1=259.0.

Example D-78[(4-Hydroxy-1-methoxymethyl-isoquinoline-3-carbonyl)-amino]-acetic acida) 4-Benzyloxy-1-methoxymethyl-isoquinoline-3-carboxylic acid benzylester

To a solution of 1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid (670mg, 2.5 mmol; Example D-77 a) in anhydrous THF (100 ml) was added slowlya 2.5 M solution of n-BuLi in hexanes (4 ml, 10 mmol) at −78° C. withstirring. After stirring for another 5 min MeOCH₂I (446 μl, 5 mmol) wasadded. Stirring was continued for additional 5 min at −78° C. beforewater (50 ml) and aqueous 6N HCl (2 ml) were added. The mixture wasallowed to warm up to ambient temperature with stirring, was thenconcentrated in vacuo to ca. ⅓ of its volume and extracted with EtOAc(50 ml). The organic phase was washed with a solution of sodiummetabisulfite (0.5 g) in water (10 ml), then dried over MgSO₄ andconcentrated in vacuo to give a yellowish solid (432 mg). A mixture of429 mg of the aforementioned yellowish solid, benzyl bromide (0.6 ml, 5mmol), K₂CO₃ (2.07 g, 15 mmol) and acetone (40 ml) was refluxed withstirring for 2.5 d. Then the mixture was concentrated in vacuo. To theresidue was added water (40 ml) and the mixture was extracted with EtOAc(50 ml). The organic phase was dried over MgSO₄ and concentrated invacuo to give a brown oil. Purification by flash column chromatographyon silica gel using hexanes:EtOAc=6:4 as the eluent gave the titlecompound as yellow oil (201 mg); MS-(+)-ion: M+1=414.1.

b) 4-Benzyloxy-1-methoxymethyl-isoquinoline-3-carboxylic acid

A mixture of 4-Benzyloxy-1-methoxymethyl-isoquinoline-3-carboxylic acidbenzyl ester (198 mg, 0.48 mmol), KOH (325 mg, 5 mmol) and EtOH (10 ml)was stirred at ambient temperature for 18 h before the solvent wasevaporated in vacuo. To the residue was added water (25 ml) and themixture was washed with Et₂O (2×25 ml). Then the solution was acidifiedby addition of aqueous 6N HCl and extracted with EtOAc (25 ml). Theorganic phase was dried over MgSO₄ and concentrated in vacuo to give thetitle compound as a yellow oil (140 mg); MS-(+)-ion: M+1=324.1.

c) [(4-Benzyloxy-1-methoxymethyl-isoquinoline-3-carbonyl)-amino]-aceticacid benzyl ester

To a mixture of 4-benzyloxy-1-methoxymethyl-isoquinoline-3-carboxylicacid (120 mg, 0.37 mmol), NEt₃ (109 μl, 0.78 mmol), and CH₂Cl₂ (7 ml)cooled with an ice bath was added ClCO₂iBu (52 μl, 0.39 mmol) withstirring. After stirring for 15 min glycine benzyl ester hydrochloride(79 mg, 0.39 mmol) was added and the mixture was stirred for another 15min before the ice bath was removed. Stirring was then continued atambient temperature for additional 1.5 h. Subsequently the mixture wasconcentrated in vacuo. To the residue was added water (10 ml) and a fewdrops of aqueous 6N HCl. The mixture was extracted with EtOAc (15 ml).The organic phase was dried over MgSO₄ and concentrated in vacuo to givea yellowish gum. Purification by flash column chromatography on silicagel using hexanes:EtOAc=7:3 as the eluent gave the title compound as ayellow oil (141 mg); MS-(+)-ion: M+1=471.1.

d) [(4-Hydroxy-1-methoxymethyl-isoquinoline-3-carbonyl)-amino]-aceticacid

A mixture of[(4-benzyloxy-1-methoxymethyl-isoquinoline-3-carbonyl)-amino]-aceticacid benzyl ester (134 mg, 0.285 mmol), Pd/C (100 mg, 10 wt % Pd), EtOAc(10 ml) and MeOH (50 ml) was stirred under a H₂-atmosphere at ambientpressure and temperature for 18 h. Then the mixture was filtered througha pad of celite. Celite and filter cake were washed thoroughly withEtOAc and the combined organic phases were concentrated in vacuo to givethe title compound as a tan solid (74 mg); MS-(−)-ion: M−1=289.2.

Example D-79[(1-Dimethylcarbamoyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 4-Benzyloxy-1-dimethylcarbamoyl-isoquinoline-3-carboxylic acidbenzyl ester

To a solution of 1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid (670mg, 2.5 mmol; Example D-77 a) in anhydrous THF (100 ml) was added slowlya 2.5 M solution of n-BuLi in hexanes (4 ml, 10 mmol) at −78° C. withstirring. After stirring for another 5 min ClCONMe₂ (468 μl, 5 mmol) wasadded. Stirring was continued for additional 25 min at −78° C. beforewater (50 ml) and aqueous 6N HCl (2 ml) were added. The mixture wasallowed to warm up to ambient temperature with stirring, was thenconcentrated in vacuo to ca. ⅓ of its volume and extracted with EtOAc(2×50 ml). The combined organic phases were dried over MgSO₄ andconcentrated in vacuo to give a yellow solid (501 mg). A mixture of 492mg of the aforementioned yellow solid, benzyl bromide (0.6 ml, 5 mmol),K₂CO₃ (2.07 g, 15 mmol) and acetone (40 ml) was refluxed with stirringfor 2.5 days. Then the mixture was concentrated in vacuo. To the residuewas added water (20 ml) and the mixture was extracted with EtOAc (50ml). The organic phase was dried over MgSO₄ and concentrated in vacuo togive a brown oil. Purification by flash column chromatography on silicagel using hexanes:EtOAc=6:4 as the eluent gave the title compound asyellow oil (311 mg); MS-(+)-ion: M+1=441.1.

b) 4-Benzyloxy-1-dimethylcarbamoyl-isoquinoline-3-carboxylic acid

A mixture of 4-benzyloxy-1-dimethylcarbamoyl-isoquinoline-3-carboxylicacid benzyl ester (308 mg, 0.7 mmol), KOH (325 mg, 5 mmol) and EtOH (10ml) was stirred at ambient temperature for 18 h before the solvent wasevaporated in vacuo. To the residue was added water (25 ml) and themixture was washed with Et₂O (2×25 ml). Then the solution was acidifiedby addition of aqueous 6N HCl and extracted with EtOAc (2×25 ml). Thecombined organic phases were dried over MgSO₄ and concentrated in vacuoto give the title compound as a yellowish gum (220 mg); MS-(+)-ion:M+1=351.0.

c)[(4-Benzyloxy-1-dimethylcarbamoyl-isoquinoline-3-carbonyl)-amino]-aceticacid benzyl ester

To a mixture of4-benzyloxy-1-dimethylcarbamoyl-isoquinoline-3-carboxylic acid (210 mg,0.6 mmol), NEt₃ (175 μl, 1.25 mmol), and CH₂Cl₂ (12 ml) cooled with anice bath was added ClCO₂iBu (83 μl, 0.63 mmol) with stirring. Afterstirring for 15 min glycine benzyl ester hydrochloride (127 mg, 0.63mmol) was added and the mixture was stirred for another 15 min beforethe ice bath was removed. Stirring was then continued at ambienttemperature for additional 1.5 h. Subsequently; the mixture wasconcentrated in vacuo. To the residue was added water (10 ml) and a fewdrops of aqueous 6N HCl. The mixture was extracted with EtOAc (15 ml).The organic phase was dried over MgSO₄ and concentrated in vacuo to givea yellowish gum. Purification by flash column chromatography on silicagel using hexanes:EtOAc=7:3 as the eluent gave the title compound as aslightly yellowish gum (211 mg); MS-(+)-ion: M+1=498.1.

d)[(1-Dimethylcarbamoyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

A mixture of[(4-Benzyloxy-1-dimethylcarbamoyl-isoquinoline-3-carbonyl)-amino]-aceticacid benzyl ester (209 mg, 0.42 mmol), Pd/C (100 mg, 10 wt % Pd), EtOAc(10 ml) and MeOH (50 ml) was stirred under a H₂-atmosphere at ambientpressure and temperature for 18 h. Then the mixture was filtered througha pad of celite. Celite and filter cake were washed thoroughly withEtOAc and the combined organic phases were concentrated in vacuo to givethe title compound as a brown solid (122 mg); MS-(−)-ion: M−1=316.1.

Example D-80[(4-Hydroxy-1-methyl-6-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 1-Bromo-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylic acid

Synthesized from 1-bromo-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylicacid butyl ester (Example D-8 a) in analogy to Example D-77 a); ¹H NMR(DMSO-d6): δ=8.20 (d, 1H), 7.21 to 7.74 (m, 7H).

b) 4-Benzyloxy-1-methyl-6-phenoxy-isoquinoline-3-carboxylic acid benzylester

To a solution of 1-Bromo-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylicacid (721 mg, 2 mmol) in anhydrous THF (100 ml) was added slowly a 2.5 Msolution of n-BuLi in hexanes (3.2 ml, 8 mmol) at −78° C. with stirring.After stirring for another 10 min MeI (253 μl, 4 mmol) was addeddropwise. Stirring was continued for additional 15 min at −78° C. beforewater (50 ml) and aqueous 2N HCl (5 ml) were added. The mixture wasallowed to warm up to ambient temperature with stirring, was thenconcentrated in vacuo to ca. ⅓ of its volume. The precipitate formed wassucked off, washed with water, and dried in vacuo to give a tan solid(758 mg). A mixture of 738 mg of the aforementioned tan solid, benzylbromide (1.0 ml, 8 mmol), K₂CO₃ (2.76 g, 20 mmol) and acetone (50 ml)was refluxed with stirring for 3 days. Then the mixture was concentratedin vacuo. To the residue was added water (30 ml) and the mixture wasextracted with EtOAc (50 ml). The organic phase was dried over MgSO₄ andconcentrated in vacuo to give a yellowish oil. Purification by flashcolumn chromatography on silica gel using hexanes:EtOAc=8:2 as theeluent gave a tan solid. Recrystallization from MeOH gave the titlecompound as slightly yellowish solid (172 mg); MS-(+)-ion: M+1=476.1.

c) 4-Benzyloxy-1-methyl-6-phenoxy-isoquinoline-3-carboxylic acid

Synthesized from4-benzyloxy-1-methyl-6-phenoxy-isoquinoline-3-carboxylic acid benzylester in analogy to Example D-78 b); MS-(+)-ion: M+1=386.1.

d)[(4-Benzyloxy-1-methyl-6-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid benzyl ester

Synthesized from4-benzyloxy-1-methyl-6-phenoxy-isoquinoline-3-carboxylic acid in analogyto Example D-78 c); MS-(+)-ion: M+1=533.0.

e) [(4-Hydroxy-1-methyl-6-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from[(4-benzyloxy-1-methyl-6-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid benzyl ester in analogy to Example D-78 d); MS-(+)-ion: M+1=353.1.

Example D-81[(4-Hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid

Synthesized from 1-bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylicacid butyl ester (Example D-7 e) in analogy to Example D-77 a);MS-(+)-ion: M+1=359.9.

b) 4-Benzyloxy-1-methyl-7-phenoxy-isoquinoline-3-carboxylic acid benzylester

Synthesized from MeI and1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid in analogy toExample D-78 a); MS-(+)-ion: M+1=476.1.

c) 4-Benzyloxy-1-methyl-7-phenoxy-isoquinoline-3-carboxylic acid

Synthesized from4-benzyloxy-1-methyl-7-phenoxy-isoquinoline-3-carboxylic acid benzylester in analogy to Example D-78 b); MS-(+)-ion: M+1=386.0.

d)[(4-Benzyloxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid benzyl ester

Synthesized from4-benzyloxy-1-methyl-7-phenoxy-isoquinoline-3-carboxylic acid in analogyto Example D-78 c); MS-(+)-ion: M+1=533.0.

e) [(4-Hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from[(4-benzyloxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid benzyl ester in analogy to Example D-78 d); MS-(−)-ion: M−1=351.1.

Example D-82[(4-Benzyloxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

A mixture of[(4-benzyloxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid benzyl ester (160 mg, 0.3 mmol; Example D-81 d), KOH (325 mg, 5mmol) and EtOH (10 ml) was stirred at ambient temperature for 18 hbefore the solvent was evaporated in vacuo. To the residue was addedwater (5 ml) and the mixture was washed with Et₂O (2×20 ml). Then thesolution was acidified by addition of aqueous 6N HCl and extracted withEtOAc (2×20 ml). The combined organic phases were dried over MgSO₄ andconcentrated in vacuo to give the title compound as a tan gum (93 mg);MS-(+)-ion: M+1=443.0.

Example D-83[(4-Ethoxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 4-Ethoxy-1-methyl-7-phenoxy-isoquinoline-3-carboxylic acid ethylester

To a solution of 1-bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylicacid (721 mg, 2 mmol, Example D-81 a) in anhydrous THF (100 ml) wasadded slowly a 2.5 M solution of n-BuLi in hexanes (3.2 ml, 8 mmol) at−78° C. with stirring. After stirring for another 5 min MeI (253 μl, 4mmol) was added dropwise. Stirring was continued for additional 15 minat −78° C. before water (100 ml) and aqueous 2N HCl (5 ml) were added.The mixture was allowed to warm up to ambient temperature with stirring,was then concentrated in vacuo to ca. ½ of its volume and extracted withEtOAc (300 ml). The organic phase was dried over MgSO₄ and concentratedin vacuo to give an orange solid (462 mg). A mixture of 440 mg of theaforementioned orange solid, EtI (0.61 ml, 7.5 mmol), K₂CO₃ (3.0 g, 21.7mmol) and acetone (45 ml) was refluxed with stirring for 16 h. Then themixture was concentrated in vacuo. To the residue was added water (30ml) and the mixture was extracted with EtOAc (2×50 ml). The combinedorganic phases were dried over MgSO₄ and concentrated in vacuo to give abrown oil. Purification by flash column chromatography on silica gelusing hexanes:EtOAc=8:2 as the eluent gave the title compound asyellowish oil (34 mg); ¹H NMR (CDCl₃): δ=8.22 (d, 1H), 7.07 to 7.50 (m,7H), 4.50 (q, 2H), 4.20 (q, 2H), 2.80 (s, 3H), 1.43 to 1.58 (m, 6H).

b) 4-Ethoxy-1-methyl-7-phenoxy-isoquinoline-3-carboxylic acid

Synthesized from 4-ethoxy-1-methyl-7-phenoxy-isoquinoline-3-carboxylicacid ethyl ester in analogy to Example D-78 b); MS-(+)-ion: M+1=324.1.

c) [(4-Ethoxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid tert-butyl ester

Synthesized from glycine tert-butyl ester hydrochloride and4-ethoxy-1-methyl-7-phenoxy-isoquinoline-3-carboxylic acid in analogy toExample D-78 c); MS-(+)-ion: M+1=437.1.

d) [(4-Ethoxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

A mixture of[(4-ethoxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid tert-butyl ester (14 mg, 0.032 mmol) and trifluoroacetic acid (2ml) was stirred at ambient temperature for 3 h. Then the mixture wasconcentrated in vacuo and the residue dissolved in EtOH (5 ml). Themixture was evaporated in vacuo to give the title compound as ayellowish solid (12 mg); MS-(−)-ion: M−1=381.1.

Example D-84[(1-Dimethylcarbamoyl-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a)4-Benzyloxy-1-dimethylcarbamoyl-7-phenoxy-isoquinoline-3-carboxylic acidbenzyl ester

Synthesized from 1-bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylicacid (Example D-81 a) in analogy to Example D-79 a (6 eq of ClCONMe₂were used, reaction mixture was stirred at −78° C. for 75 min after theaddition of ClCONMe₂ was finished before adding water and HCl);MS-(+)-ion: M+1=533.2.

b) 4-Benzyloxy-1-dimethylcarbamoyl-7-phenoxy-isoquinoline-3-carboxylicacid

Synthesized from4-benzyloxy-1-dimethylcarbamoyl-7-phenoxy-isoquinoline-3-carboxylic acidbenzyl ester in analogy to Example D-79 b); MS-(−)-ion: M−1=441.1.

c)[(4-Benzyloxy-1-dimethylcarbamoyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid benzyl ester

Synthesized from4-benzyloxy-1-dimethylcarbamoyl-7-phenoxy-isoquinoline-3-carboxylic acidin analogy to Example D-79 c); MS-(+)-ion: M+1=590.0.

d)[(1-Dimethylcarbamoyl-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from[(4-benzyloxy-1-dimethylcarbamoyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid benzyl ester in analogy to Example D-79 d); MS-(+)-ion: M+1=410.0.

Example D-85[(4-Hydroxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 4-Benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carboxylicacid benzyl ester

Synthesized from 1-bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylicacid (Example D-81 a) in analogy to Example D-78 a); MS-(+)-ion:M+1=506.2.

b) 4-Benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carboxylic acid

Synthesized from4-Benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carboxylic acidbenzyl ester in analogy to Example D-78 b); MS-(−)-ion: M−1=414.1.

c)[(4-Benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid benzyl ester

Synthesized from4-benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carboxylic acid inanalogy to Example D-78 c); MS-(+)-ion: M+1=563.1.

d)[(4-Hydroxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized[(4-benzyloxy-1-methoxymethyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid benzyl ester in analogy to Example D-78 d); MS-(+)-ion: M+1=383.0.

Example D-86[(4-Hydroxy-1-p-tolyl-isoquinoline-3-carbonyl)-amino]-acetic acid a)4-Benzyloxy-1-bromo-isoquinoline-3-carboxylic acid butyl ester

A mixture of 1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid butylester (6.48 g, 20 mmol; Example D-28 a), benzyl bromide (3.6 ml, 30mmol), K₂CO₃ (12.44 g, 90 mmol) and acetone (300 ml) was refluxed withstirring for 2.5 d. The solvent was then evaporated in vacuo. To theresidue was added water (100 ml) and the mixture was extracted withEtOAc (100 ml). The organic phase was dried over MgSO₄ and evaporated invacuo to give the title compound as a yellowish solid; MS-(+)-ion:M+1=414.1.

b) 4-Benzyloxy-1-p-tolyl-isoquinoline-3-carboxylic acid butyl ester

4-Benzyloxy-1-bromo-isoquinoline-3-carboxylic acid butyl ester (207 mg,0.5 mmol) and Pd(PPh₃)₄ (23 mg, 0.02 mmol) were dissolved in THF (3 ml)and the solution was stirred for 10 min before a solution ofp-tolylboronic acid (68 mg, 0.5 mmol) in EtOH (0.5 ml) and a solution ofNa₂CO₃ (106 mg, 1 mmol) in water (0.5 ml) were added. The resultingmixture was refluxed with stirring for 4 h. Subsequently, the mixturewas concentrated in vacuo. To the residue was added water (2 ml) and themixture was extracted with EtOAc (10 ml). The organic phase was driedover MgSO₄ and evaporated in vacuo to give a yellowish oil (225 mg).Purification by flash column chromatography on silica gel usinghexanes:EtOAc=94:6 as the eluent gave the title compound as a colorlessoil; MS-(+)-ion: M+1=426.2.

c) 4-Hydroxy-1-p-tolyl-isoquinoline-3-carboxylic acid butyl ester

Synthesized from (4-benzyloxy-1-p-tolyl-isoquinoline-3-carboxylic acidbutyl ester in analogy to Example D-78 d) (EtOAc was used as thesolvent); MS-(+)-ion: M+1=336.2.

d) [(4-Hydroxy-1-p-tolyl-isoquinoline-3-carbonyl)-amino]-acetic acid

Synthesized from 4-hydroxy-1-p-tolyl-isoquinoline-3-carboxylic acidbutyl ester in analogy to Example D-1 g); MS-(+)-ion: M+1=337.1.

Example D-87{[7-(4-Fluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-aceticacid a) 1-Bromo-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Synthesized from7-(4-fluoro-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acid butylester (Example D-96 e) in analogy to Example D-1 e); ¹H NMR (CDCl₃):δ=11.89 (s, 1 H), 8.36 (d, 1H), 7.44 to 7.57 (m, 2H), 7.08 to 7.25 (m,4H), 4.47 (q, 2H), 1.85 (m, 2H), 1.50 (m, 2H), 0.99 (t, 3H).

b) 7-(4-Fluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylicacid butyl ester

A mixture of1-bromo-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (434 mg, 1 mmol), Pd(PPh₃)₄ (116 mg, 0.1 mmol),trimethylboroxine (140 μl, 1 mmol), K₂CO₃ (414 mg, 3 mmol), and1,4-dioxane (8 ml) was refluxed with stirring for 2 h. Subsequently, themixture was concentrated in vacuo. To the residue was added water (10ml). The mixture was acidified by the addition of aqueous 6N HCl andthen extracted with EtOAc (40 ml). The organic phase was dried overMgSO₄ and evaporated in vacuo. Purification of the residue by flashcolumn chromatography on silica gel using hexanes:EtOAc=94:6 as theeluent gave the title compound as white solid (229 mg); MS-(+)-ion:M+1=370.1.

c){[7-(4-Fluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-aceticacid

Synthesized from7-(4-fluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylic acidbutyl ester in analogy to Example D-1 g); MS-(+)-ion: M+1=371.1.

The above method can be used to synthesize other intermediates usedherein.

Example D-88{[1-Chloro-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-(4-Methoxy-phenoxy)-phthalonitrile

A mixture of 4-nitro-phthalonitrile (4.00 g), 4-methoxy-phenol (3.46 g)and potassium carbonate (6.39 g) in acetone (64 ml) was heated to refluxfor 2 h. Reaction mixture was cooled and filtered. Filtrate wasconcentrated and the residue was dissolved in ethyl acetate (100 ml).The solution was washed with NaOH (1 N, 50 ml), water, and then brine.The organic layer was dried over magnesium sulfate, filtered, andconcentrated to give the product (6.14 g). ¹H NMR (200 MHz, CDCl₃) δ6.70 (d, J=7.8 Hz, 1H), 7.21 (m, 2H), 6.96 (m, 4H), 3.84 (s, 3H).

b) 4-(4-Methoxy-phenoxy)-phthalic acid

Prepared in analogy to Example D-1 a). MS-(−)-ion: M−1=286.9.

c) [5-(4-Methoxy-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

Prepared in analogy to Example D-37 b). ¹H NMR (200 MHz, CDCl₃) δ 7.74(d, J=8.6 Hz, 1H), 7.25 (m, 2H), 6.98 (m, 4H), 4.40 (s, 2H), 3.83 (s,3H), 3.75 (s, 3H).

d) 6- and 7-(4-Methoxy-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-21 b). MS-(+)-ion: M+1=384.10.

e) 7-(4-Methoxy-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example D-21 c). MS-(+)-ion: M+1=384.11.

f) 1-Chloro-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-43 d). MS-(+)-ion: M+1=402.0.

g){[1-Chloro-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-1 g). MS-(−)-ion: M−1=400.96.

Example D-89{[4-Hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a){[4-Hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

Synthesized from{[1-chloro-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid in analogy to Example D-25. MS-(−)-ion: M−1=367.0.

Example D-90{[1-Chloro-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 6-(4-Methoxy-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

Separate from the mixtures of 6- and7-(4-methoxy-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acid butylester obtained from Example D-88 e). MS-(+)-ion: M+1=384.1

b) 1-Chloro-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-43 d). MS-(+)-ion: M+1=402.0.

c){[1-Chloro-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-1 g). MS-(+)-ion: M+1=403.0.

Example D-91{[4-Hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-2 a) from{[1-chloro-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid. MS-(−)-ion: M−1=367.0.

The compounds of Example D-92-99 below were obtained by processanalogous to those described in Examples D88-D91.

Example D-92{[1-Chloro-4-hydroxy-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-(4-Trifluoromethyl-phenoxy)-phthalonitrile

¹H NMR (200 MHz, CDCl₃) δ 7.74 (m, 2H), 7.47 (d, J=8.6 Hz, 1H), 7.25 (m,3H), 6.87 (d, J=8.9 Hz, 1H).

b) 4-(4-Trifluoromethyl-phenoxy)-phthalic acid

¹H NMR (200 MHz, DMSO-d6) δ 8.24 (d, J=9.0 Hz, 1H), 7.75 (m, 3H), 7.19(m, 3H)

c)[1,3-Dioxo-5-(4-trifluoromethyl-phenoxy)-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

¹H NMR (200 MHz, CDCl₃) δ 7.86 (d, J=8.5 Hz, 1H), 7.67 (d, J=8.2 Hz,2H), 7.40-7.13 (m, 4H), 4.43 (s, 2H), 3.76 9 s, 3H)

d) 6- and7-(4-trifluoromethyl-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

Mixture of two isomers.

e) 7-(4-trifluoromethyl-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

MS-(+)-ion: M+1=422.0

f)1-Chloro-4-hydroxy-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

MS-(−)-ion: M−1=438.3

g){[1-Chloro-4-hydroxy-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

MS-(−)-ion: M−1=439.0.

Example D-93{[4-Hydroxy-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

MS-(−)-ion: M−1=405.1

Example D-94{[1-Chloro-4-hydroxy-6-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a)1,4-Dihydroxy-6-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

MS-(+)-ion: M+1=422.0

b)1-Chloro-4-hydroxy-6-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

¹H NMR (200 MHz, CDCl₃) δ 11.82 (s, 1H), 8.30 (d, J=9.0 Hz, 1H), 7.81(d, J=2.3 Hz, 1H), 7.67 (d, J=8.6 Hz, 2H), 7.54 (dd, J=9.0, 2.7 Hz, 1H),7.18 (d, J=8.2 Hz, 2H), 4.48 (t, J=7.0 Hz, 2H), 1.85 (m, 2H), 1.46 (m,2H), 0.98 (t, J=7.0 Hz, 3H).

c){[1-Chloro-4-hydroxy-6-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

MS-(−)-ion: M−1=439.1.

Example D-95{[4-Hydroxy-6-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

MS-(−)-ion: M−1=405.0.

Example D-96{[1-Chloro-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-(4-Fluoro-phenoxy)-phthalonitrile

¹H NMR (200 MHz, CDCl₃) δ 7.71 (d, J=8.6 Hz, 1H), 7.23-7.15 (m, 6H).

b) 4-(4-Fluoro-phenoxy)-phthalic acid

¹H NMR (200 MHz, CDCl₃) δ 7.74 (d, J=8.9 Hz, 1H), 7.33-7.15 (m, 6H).

c) [5-(4-Fluoro-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

¹H NMR (200 MHz, CDCl₃) δ 7.80 (d, J=7.4 Hz, 1H), 7.28 (m, 2H), 7.08 (m,4H), 4.41 (s, 2H), 3.76 (s, 3H).

d) 6- and 7-(4-Fluoro-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

A mixture of two isomers.

e) 7-(4-Fluoro-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester

MS-(+)-ion: M+1=372.1

f) 1-Chloro-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

¹H NMR (200 MHz, CDCl₃) δ 11.90 (s, 1H), 8.36 (d, J=9.0 Hz, 1H), 7.56(m, 2H), 7.10 (m, 4H), 4.47 (t, J=7.0 Hz, 2H), 1.85 (m, 2H), 1.46 (m,2H), 0.99 (t, J=7.4 Hz, 3H).

g){[1-Chloro-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

MS-(−)-ion: M−1=389.0.

Example D-97{[7-(4-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

MS-(−)-ion: M−1=355.1.

Example D-98{[1-Chloro-6-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a) 6-(4-Fluoro-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

MS-(+)-ion: M+1=372.1

b) 1-Chloro-6-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

¹H NMR (200 MHz, CDCl₃) δ 11.77 (s, 1H), 8.25 (d, J=9.0 Hz, 1H), 7.62(d, J=2.3 Hz, 1H), 7.50 (dd, J=9.0, 2.3 Hz, 1H), 7.10 (m, 4H), 4.46 (t,J=7.0 Hz, 2H), 1.85 (m, 2H), 1.45 (m, 2H), 0.98 (t, J=7.4 Hz, 3H).

c){[1-Chloro-6-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

MS-(−)-ion: M−1=389.1.

Example D-99{[6-(4-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

MS-(−)-ion: M−1=355.1.

Example D-100{[4-Hydroxy-7-(pyridin-4-ylsulfanyl)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-(Pyridin-4-ylsulfanyl)-phthalonitrile

A mixture of 4-nitro-phthalionitrile (17.28 g), pyridine-4-thiol (10.68g) and potassium carbonate (25.17 g) in N,N-dimethyl-formamide (160 ml)was heated to 85 C and stirred for 3 h. After cooling, the reactionmixture was filtered through a pad of celite and rinsed with ethylacetate. Filtrate was concentrated in vacuo and the residue was purifiedby silica gel chromatography (eluting with 15-30% of ethyl acetate inmethylene chloride) to give the title compound 13.29 g. ¹H NMR (200 MHz,CDCl₃) δ 8.59 (d, J=6.2 Hz, 2H), 7.68 (m, 3H), 7.24 (d, J=6.3 Hz, 2H).

b) 4-(Pyridin-4-ylsulfanyl)-phthalic acid

Prepared in analogy to Example D-1 a). MS-(+)-ion: M+1=276.1.

c) [1,3-Dioxo-5-(pyridin-4-ylsulfanyl)-1,3-dihydro-isoindol-2-yl]-aceticacid butyl ester

A solid mixture of 4-(pyridin-4-ylsulfanyl)-phthalic acid (11.40 g) andglycine n-butyl ester hydrochloride salt (6.95 g) was heated in a oilbath (250° C.) with efficient stirring for 20 min. until the waterbubble evaporation ceased. After cooling, it was partitioned betweenethyl acetate (300 ml) and saturated sodium bicarbonate aqueous solution(150 ml). Two layers were separated and the aqueous layer was extractedwith ethyl acetate (300 ml). Combined organic layers were washed withbrine, dried over magnesium sulfate, filtered and concentrated to givethe title compound 10.70 g. MS-(+)-ion: M+1=371.2.

d) 6- and7-(Pyridin-4-ylsulfanyl)-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example D-21 b).

e) 6- and7-(Pyridin-4-ylsulfanyl)-1-chloro-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-43 d). MS-(+)-ion: M+1=389.1.

f) 6- and 7-(Pyridin-4-ylsulfanyl)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-1 f). Crude product was purified bysilica gel chromatography (50%-80% ethyl acetate in methylene chloride)to give7-(pyridin-4-ylsulfanyl)-1-chloro-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound D-100A) (MS-(+)-ion: M+1=355.04) and6-(pyridin-4-ylsulfanyl)-1-chloro-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound D-100B) (MS-(+)-ion: M+1=355.13).

g){[4-Hydroxy-7-(pyridin-4-ylsulfanyl)-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-1 g). MS-(+)-ion: M+1=356.1.

Example D-101{[4-Hydroxy-6-(pyridin-4-ylsulfanyl)-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-1 g) from6-(pyridin-4-ylsulfanyl)-1-chloro-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound D-100 B). MS-(+)-ion: M+1=356.1.

Example D-102[(7-Benzenesulfinyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 7-Benzenesulfinyl-4-hydroxy-isoquinoline-3-carboxylic acid butylester

A slurry mixture of 4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester (300 mg) and OXONE® Dupont Specialty Chemicals,Willmington, Del., USA) (366 mg) in (3/2) methanol/water (5 ml) wasstirred at room temp for 4 h. Reaction mixture was partitioned betweenmethylene chloride and saturated aqueous sodium bicarbonate solution.Organic layer was washed with saturated aqueous sodium bicarbonatesolution and water. Dried over magnesium sulfate and filtered. Filtratewas concentrated and the residue was purified by silica gelchromatograph (0%-50% ethyl acetate in methylene chloride) to give thetitle compound 7-benzenesulfinyl-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound D-102 A) (50 mg) (MS-(+)-ion: M+1=370.1) and7-benzenesulfonyl-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound D-102B) (90 mg) (MS-(+)-ion: M+1=386.1).

b) [(7-Benzenesulfinyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared in analogy to Example D-1 g) from7-benzenesulfinyl-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound D-102 A). MS-(+)-ion: M+1=371.1.

Example D-103[(7-Benzenesulfonyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared in analogy to Example D-1 g) from7-benzenesulfonyl-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound D-102 B). MS-(+)-ion: M+1=387.1.

Example D-104[(6-Benzenesulfinyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 6-Benzenesulfinyl-4-hydroxy-isoquinoline-3-carboxylic acid butylester

Prepared in analogy to Example D-18 a) from4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carboxylic acid butyl ester.Two compounds were isolated from chromatography: the title compound6-benzenesulfinyl-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound D-104 A) (MS-(+)-ion: M+1=370.1) and6-benzenesulfonyl-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound D-104 B) (MS-(+)-ion: M+1=386.1).

b) [(6-Benzenesulfinyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared in analogy to Example D-1 g) from6-benzenesulfinyl-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound D-104 A). MS-(−)-ion: M−1=369.0.

Example D-105[(6-Benzenesulfonyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared in analogy to Example D-1 g) from6-benzenesulfonyl-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound D-104 B). MS-(−)-ion: M−1=385.1.

Example D-106 [(6-Amino-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) (5-Nitro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid ethylester

Potassium carbonate (57.8 g) was added to a solution mixture of5-nitro-isoindole-1,3-dione (26.2 g) and bromo-acetic acid ethyl ester(25.1 g) in acetone (500 ml). The resulting mixture was refluxedovernight (18 h). After cooling, reaction mixture was filtered andrinsed with ethyl acetate. Filtrate was concentrated and the residue wastriturated with ether (200 ml). Solid was collected and rinsed withether. Dried in vacuo to give the title compound 231.9 g. ¹H NMR (200MHz, CDCl₃) δ 8.69 (m, 2H), 8.07 (d, J=8.2 Hz, 1H), 4.48 (s, 2H), 4.24(q, J=7.0 Hz, 2H), 1.30 (t, J=7.0 Hz, 3H).

b) (5-Amino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid ethyl ester

10% Palladium/C (50% wet) solid (2.0 g) was added to a solution mixtureof (5-nitro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid ethyl ester(10.0 g) in glacial acetic acid (150 ml). Stirred vigorously under H₂(balloon pressure) at room temperature overnight (18 h). Catalyst wasfiltered off through a pad of celite and rinsed with methylene chloride.Filtrate was concentrated to give the title compound (7.0 g). ¹H NMR(200 MHz, CDCl₃) δ 7.59 (d, J=8.2 Hz, 1H), 7.02 (d, J=2.0 Hz, 1H), 6.81(dd, J=8.2, 2.0 Hz, 1H), 4.38 (br S, 2H), 4.36 (s, 2H), 4.20 (q, J=7.0Hz, 2H), 1.27 (t, J=7.0 Hz, 3H).

c)[5-(Benzhydrylidene-amino)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester

Titanium tetrachloride (1.99 g) was slowly added to a mixture of(5-amino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid ethyl ester(3.48 g), benzophenone (2.81 g) and DABCO (4.72 g) in chlorobenzene (112ml). Resulting mixture was heated to reflux for 2.5 h. After cooling,reaction mixture was filtered through a pad of celite and rinsed withethyl acetate. Filtrate was concentrated and the residue was purified bysilica gel chromatography (25%-40% ethyl acetate in hexanes) to give thetitle compound (3.03 g). MS-(+)-ion: M+1=413.3.

d) 6- and7-(Benzhydrylidene-amino)-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example D-21 b). MS-(+)-ion: M+1=441.2

e) 6- and 7-Amino-1-chloro-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example D-43 d). The crude product was purifiedby silica gel chromatography (eluting with 50% ethyl acetate in hexanes)to give the title compounds. MS-(+)-ion: M+1=295.1.

f) 6- and 7-Amino-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

To a solution of 6- and7-Amino-1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester(220 mg) in ethyl acetate (5 ml) was added 10% Pd/C (50% wet) (110 mg)and then ammonium formate (471 mg). Resulting mixture was heated toreflux for 0.5 h. After cooling, the reaction mixture was diluted withethyl acetate (50 ml) and filtered. Filtrate was concentrated to givethe title compounds 182 mg. MS-(+)-ion: M+1=261.2.

g) 6- and7-Amino-4-(4-methoxy-benzenesulfonyloxy)-isoquinoline-3-carboxylic acidbutyl ester

A mixture of 6- and 7-amino-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (180 mg), 4-methoxy-benzenesulfonyl chloride (145 mg) andtriethyl amine (85 mg) in methylene chloride (7 ml) was stirred at roomtemperature for 18 h. It was diluted with water (20 ml) and acidified topH 4 by 0.1 N HCl aqueous solution. Two phases were separated and theaqueous layer was extracted with methylene chloride. Combined organiclayers were washed with brine, dried over magnesium sulfate, filteredand concentrated. Crude product was purified by silica gelchromatography (55%-80% ethyl acetate in hexanes) to two products:7-amino-4-(4-methoxy-benzenesulfonyloxy)-isoquinoline-3-carboxylic acidbutyl ester (Compound D-106 A) (79 mg) (MS-(+)-ion: M+1=431.1) and6-amino-4-(4-methoxy-benzenesulfonyloxy)-isoquinoline-3-carboxylic acidbutyl ester (Compound D-106 B) (70 mg) (MS-(+)-ion: M+1=431.1).

h) [(6-Amino-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid

Prepared from6-amino-4-(4-methoxy-benzenesulfonyloxy)-isoquinoline-3-carboxylic acidbutyl ester (Compound D-106 B) in analogy to Example D-1 g). MS-(−)-ion:M−1=260.1.

Example D-107{[4-Hydroxy-7-(4-methoxy-benzenesulfonylamino)-isoquinoline-3-carbonyl]-amino}-aceticacid a)7-[(N,N-Di-4-methoxy-benzenesulfonyl)amino]-4-(4-methoxy-benzenesulfonyloxy)-isoquinoline-3-carboxylicacid butyl ester

A mixture of7-amino-4-(4-methoxy-benzenesulfonyloxy)-isoquinoline-3-carboxylic acidbutyl ester (Compound D-106 A) (75 mg), 4-methoxy-benzenesulfonylchloride (140 mg) and triethyl amine (76 mg) in methylene chloride (2ml) in a sealed vessel was heated in a microwave reactor to 120 C for 10min. After cooling, reaction mixture was concentrated and purified bysilica gel chromatography (eluting with 5%-10% ethyl acetate inmethylene chloride) to give the title compound (68 mg). MS-(+)-ion:M+1=770.99.

b){[4-Hydroxy-7-(4-methoxy-benzenesulfonylamino)-isoquinoline-3-carbonyl]-amino}-aceticacid

A mixture of the above ester (68 mg) and glycine (86 mg) in 0.5 N sodiummethoxide/methanol (2.7 ml) in a sealed vessel was heated in a microwavereactor (150° C., 17 min). After cooling, reaction mixture wasconcentrated. Residue was dissolved in water (10 ml) and extracted withethyl acetate (15 ml). Aqueous layer was acidified by 2 N HCl aqueoussolution to pH=4 and extracted with ethyl acetate (2×50 ml). Combinedorganic layers were washed with brine, dried over magnesium sulfate,filtered, and concentrated. The crude product was triturated withmethanol and (1/1) ethyl acetate/hexanes to give the title compound. 14mg. MS-(−)-ion: M−1=430.

Example D-108{[4-Hydroxy-7-(3-phenyl-ureido)-isoquinoline-3-carbonyl]amino}-aceticacid a) 6- and 7-(3-Phenyl-ureido)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

A mixture of 6- and 7-Amino-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (160 mg) and phenyl isocyanate (73 mg) in methylene chloride(4 ml) was stirred at room temperature overnight (18 h) andconcentrated. Residue was triturated with (1/1) ethyl acetate/methylenechloride (8 ml). Insoluble solid was collected by filtration and rinsedwith methylene chloride (5 ml). It was dried to give7-(3-phenyl-ureido)-4-Hydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound D-108 A) (82 mg) (MS-(+)-ion: M+1=380.18). Filtrate wasconcentrated and the residue was purified by silica gel chromatographyand then recrystallized from methanol to give6-(3-phenyl-ureido)-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound D-108 B) (82 mg) (MS-(+)-ion: M+1=380.15).

b){[4-Hydroxy-7-(3-phenyl-ureido)-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared from 7-(3-phenyl-ureido)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound D-108 A) in analogy to Example D-1 g).MS-(−)-ion: M−1=379.07.

Example D-109{[4-Hydroxy-6-(3-phenyl-ureido)-isoquinoline-3-carbonyl]-amino}-aceticacid a){[4-Hydroxy-6-(3-phenyl-ureido)-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared from 7-(3-phenyl-ureido)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound D-108 B) in analogy to Example D-1 g).MS-(−)-ion: M−1=379.08.

Example D-110[(4-Hydroxy-1-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid

The title compound was prepared as follows: To a solution of 250 mg of[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid, (U.S.Pat. No. 6,093,730, disclosed asN-((1-chloro-4-hydroxyisoquinoline-3-yl)carbonyl)glycine), in 1 ml1-methyl-2-pryrrolidinone was added 1.2 ml of benzenethiol. The solutionwas heated at 130 to 150° C. in a sealed tube for 16 h. The solution wasconcentrated under vacuum. The resultant residue was crystallized frommethanol to yield 91 mg of a tan solid; MS (−) m/z 353.07 (M−1)

Example D-111{[1-(4-Chloro-phenylsulfanyl)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was prepared from[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid (U.S.Pat. No. 6,093,730) and 4-chlorobenzenethiol under conditions analogousto Example D-110; MS (+) m/z 389.06 (M+1)

Example D-112[(4-Hydroxy-1-p-tolylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was prepared from[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid (U.S.Pat. No. 6,093,730) and 4-methylbenzenethiol under conditions analogousto Example D-110; MS (−) m/z 367.09 (M−1)

Example D-113{[4-Hydroxy-1-(pyridin-2-ylsulfanyl)-isoquinoline-3-carbonyl]amino}-aceticacid

The title compound was prepared from[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid (U.S.Pat. No. 6,093,730) and 2-mercaptopyridine under conditions analogous toExample D-110. The final product was purified by column chromatographyon silica gel using a gradient of 3-15% methanol in dichloromethane with0.5% acetic acid to elute the product; MS (−) m/z 354.10 (M−1)

Example D-114{[4-Hydroxy-1-(3-methoxy-phenylsulfanyl)-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was prepared from[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid (U.S.Pat. No. 6,093,730) and 3-methoxybenzenethiol under conditions analogousto Example D-110. The final product was precipitated from a solution ofethyl acetate using hexanes; MS (−) m/z 385.12 (M−1)

Example D-115{[4-Hydroxy-1-(2-methoxy-phenylsulfanyl)-isoquinoline-3-carbonyl]amino}-aceticacid

The title compound was prepared from[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid (U.S.Pat. No. 6,093,730) and 2-methoxybenzenethiol under conditions analogousto Example D-110. The final product was crystallized fromdichloromethane; MS (−) m/z 383.08 (M−1)

Example D-116{[4-Hydroxy-1-(naphthalen-2-ylsulfanyl)-isoquinoline-3-carbonyl]amino}-aceticacid

The title compound was prepared from[(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid (U.S.Pat. No. 6,093,730) and 2-napthalenethiol under conditions analogous toExample D-110. The final product was purified by triturating the crudeproduct twice with methanol and twice with dichloromethane; MS (+) m/z405.08 (M+1).

Example D-117[(1-Benzenesulfinyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was prepared as follows: 50 mg of[(4-hydroxy-1-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid, Example D-110, was dissolved in 0.3 ml 1-methyl-2-pyrolydinone and0.7 ml dichloromethane. The solution was cooled to 0° C. and 26 mg of75% 3-chloroperoxybenzoic acid was added. The solution was stirred for 2hours at room temperature, then concentrated under high vacuum. Theresultant residue was triturated with ethyl acetate to provide 32 mg ofthe product as a white solid.; MS (−) m/z 369.08 (M−1)

Example D-118[(1-Benzenesulfonyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was prepared as follows: 50 mg of[(4-hydroxy-1-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid, Example D-110, was dissolved in 0.1 ml 1-methyl-2-pyrolydinone and0.7 ml dichloromethane. To the solution was added 72 mg of 75%3-chloroperoxybenzoic acid. The solution was stirred for 6 hours at roomtemperature. The mixture was partitioned between ethyl acetate andwater. The organic fraction was dried over anhydrous magnesium sulfate,and concentrated to a residue. The resultant residue was triturated withethyl acetate to provide 28 mg of the product as a white solid; MS (−)m/z 385.09 (M−1)

Example D-119{[4-Hydroxy-7-(pyridin-2-ylsulfanyl)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-(pyridin-2-ylsulfanyl)-phthalonitrile

10 g of 2-mercaptopyridine, 14.2 g of 4-nitrophthalonitrile, and 22.6 gof potassium carbonate were suspended in 250 ml of acetone and heated atreflux temperature for 4 hours. The solution was filtered through a padof celite and a course glass filter to remove residual solids. Thesolution was concentrated to a crude residue and purified by columnchromatography on silica gel eluting the product with a gradient of0-10% ethyl acetate in dichloromethane. 6.4 g of product was recovered;¹H NMR (200 Mz, CDCl₃) δ=8.49-8.53 (m, 1H), 7.84-7.83 (dd, 1H),7.76-7.71 (m, 2H), 7.68-7.64 (dd, 1H), 7.40-7.36 (dt, 1H), 7.27-7.20 (m,1H).

b){[4-Hydroxy-7-(pyridin-2-ylsulfanyl)-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was prepared from4-(pyridin-2-ylsulfanyl)-phthalonitrile in analogy to Example D-1; MS(+): m/z 356.01 (M+1).

Example D-120{[4-Hydroxy-6-(pyridin-2-ylsulfanyl)-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was prepared from4-(pyridin-2-ylsulfanyl)-phthalonitrile in analogy to Example D-119; MS(+): m/z 356.02 (M+1).

Example D-121[(1-Chloro-4-hydroxy-6,7-diphenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 4,5-diphenoxyphthalonitrile

5.0 g of 4,5-dichlorophthalonitrile was dissolved in 50 ml of DMSO. 14.3g of phenol was added and the solution was heated to 90° C. Portions of6.9 g of potassium carbonate was added every five minutes until a totalof 55.2 g had been added. The mixture was stirred at 90° C. for thirtyminutes then cooled and poured into 500 ml of ice-water. The resultingsolid precipitate was collected and crystallized from methanol toproduce 3.6 g of product; ¹H NMR (200 Mz, CDCl₃) δ=7.49-7.38 (m, 4H),7.32-7.25 (m, 2H), 7.15 (s, 2H), 7.10-7.02 (m, 4H)

b)[(1-Chloro-4-hydroxy-6,7-diphenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was synthesized from 4,5-diphenoxyphthalonitrile inanalogy to Example D-7a-d and Example D-9a-b; MS (+): m/z 465.05 (M+1).

Example D-122[(4-Hydroxy-6,7-diphenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid

The title compound was synthesized from 4,5-diphenoxyphthalonitrile,Example D-121a, in analogy to Example D-7; MS (+): m/z 431.07 (M+1).

Example D-123({4-Hydroxy-7-[4-(toluene-4-sulfonylamino)-phenoxy]-isoquinoline-3-carbonyl}-amino)-aceticacid a) 1-Chloro-4-hydroxy-6-(4-nitro-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

200 mg of 1-Chloro-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylic acidbutyl ester, Example D-10a, was dissolved in 3 ml of concentratedsulfuric acid. The reaction mixture was cooled to −20° C. and 60 mg ofpotassium nitrate was added slowly to the stirring solution. Thereaction was kept between −10 to −20° C. while stirring for 15 min, andpoured into ice-water. The aqueous mixture was extracted twice withethyl acetate. The organic fractions were washed successively withsaturated bicarbonate and brine solutions, dried over anhydrousmagnesium sulfate, and concentrated to a residue under reduced pressure.The resultant solid was triturated with ethyl acetate followed bymethanol to produce 103 mg of white solid; MS (+): m/z 417.07 (M+1).

b) 6-(4-Amino-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester

100 mg of1-chloro-4-hydroxy-6-(4-nitro-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester was dissolved in 3 ml THF and 3 ml methanol. 20 mg of sodiumacetate and 25 mg of 10% palladium on carbon were added to the mixture,and the stirring reaction was placed under hydrogen atmosphere (balloon)overnight. The resultant solution was filtered through a pad of celiteand concentrated to a residue. The crude material was purified by columnchromatography on silica gel, eluting the product with a gradient of0-20% ethyl acetate in dichloromethane, to produce 59 mg of product; MS(−): m/z 351.27 (M−1).

c)4-Hydroxy-7-[4-(toluene-4-sulfonylamino)-phenoxy]-isoquinoline-3-carboxylicacid butyl ester

58 mg of 6-(4-Amino-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester, 15.8 mg of pyridine, and 34 mg of p-toluenesulfonylchloride were dissolved in 0.3 ml of dry dichloromethane. The mixturewas stirred for 16 hours, and then partitioned between 0.25 N HCl andethyl acetate. The organic fraction was successively washed with water,saturated bicarbonate, and brine solutions, then dried over anhydroussodium sulfate, and concentrated to 84 mg of a crude solid. The crudematerial was tiriturated with ethyl acetate to produce 42 mg of a whitesolid; ¹H NMR (200 Mz, CDCl₃) δ=11.7 (s, 1H), 8.72 (d, 1H), 7.93-7.88(d, 1H), 7.69-7.65 (d, 2H), 7.56-7.54 (m, 2H), 7.44-7.39 (dd, 1H),7.27-7.13 (m, 5H), 7.00-6.96 (d, 2H), 4.46 (t, 2H), 2.4 (s, 3H),1.87-1.82 (quintet, 2H), 1.48-1.40 (quint, 2H), 1.00-0.95 (t, 3H).

d)({4-Hydroxy-7-[4-(toluene-4-sulfonylamino)-phenoxy]-isoquinoline-3-carbonyl}-amino)-aceticacid

To a solution of 1.85 ml of 0.5 M sodium methoxide in methanol was added42 mg of4-Hydroxy-7-[4-(toluene-4-sulfonylamino)-phenoxy]-isoquinoline-3-carboxylicacid butyl ester and 70 mg of glycine. The resultant mixture was heatedat reflux temperature for 24 hours and then cooled to room temperature.The reaction was poured into a 0.2 N HCl aqueous solution and thenextracted three times with ethyl acetate. The organic fractions weredried over anhydrous sodium sulfate and concentrated to 41 mg of a whitesolid; MS (+): m/z 508.10 (M+1).

Example D-124{[4-Hydroxy-7-(4-nitro-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-Hydroxy-7-(4-nitro-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester

2.0 g of 4-Hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butyl ester,Example D-7f, was dissolved in 15 ml of TFA. 0.375 ml of fuming nitricacid was added slowly to the solution, and the resultant mixture wasstirred at room temperature for 7 hours. The reaction mixture wasconcentrated under vacuum, and the resultant residue was purified bycolumn chromatography on silica gel, eluting with 0-20% ethyl acetate indichloromethane. The crude product obtained was triturated with methanolto produce 1.0 g of white solid; MS (+): m/z 383.01 (M+1).

b) {[4-Hydroxy-7-(4-nitro-phenoxy)-isoquinoline-3-carbonyl]amino}-aceticacid

The title compound was synthesized from4-hydroxy-7-(4-nitro-phenoxy)-isoquinoline-3-carboxylic acid butyl esterin analogy to Example D-1g; MS (−): m/z 382.06 (M−1).

Example D-125[(4-Mercapto-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid a)4-Dimethylthiocarbamoyloxy-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester

To a solution of 1.5 g of 4-hydroxy-7-phenoxy-isoquinoline-3-carboxylicacid butyl ester, Example D-7f, in 6.3 ml of anhydrous DMF was added 578mg of dimethylthiocarbamoylchloride and 1.5 g of1,4-diazabicyclo[2.2.2]octane. The mixture was stirred overnight at roomtemperature. The mixture was poured into 30 ml of 1 N HCl and extractedthree times with 30 ml portions of ethyl acetate. The organic fractionswere washed with water and brine, dried over anhydrous sodium sulfate,and concentrated to 1.9 g of product; MS (+) m/z 425.27 (M+1)

b) 4-Dimethylcarbamoylsulfanyl-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester

A solution of 1.9 g of4-dimethylthiocarbamoyloxy-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester in 22 ml of phenyl ether was heated to 190° C. for 2 hours.The solution was concentrated under vacuum to give a crude residue,which was purified by column chromatography on silica gel, eluting theproduct with a gradient of 30-80% ethyl acetate in hexanes to give 1.73g; MS (+) m/z 425.07 (M+1)

c) 4-Mercapto-7-phenoxy-isoquinoline-3-carboxylic acid methyl ester

To a solution of 6.5 ml of 0.5 N sodium methoxide in methanol was added460 mg of4-dimethylcarbamoylsulfanyl-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester. The resultant solution was heated to 50-60° C. for 8 hours,cooled to room temperature, and diluted with 10 ml water and 7.0 ml 1 NHCl. The resulting yellow precipitate was collected by filtering thesolution through a (medium) porous buchner filter funnel to give 307 mgof product; MS (+) m/z 312.08 (M+1)

d) [(4-Mercapto-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid

To a solution of 4.3 ml of 0.5 M sodium methoxide in methanol was added75 mg of 4-mercapto-7-phenoxy-isoquinoline-3-carboxylic acid methylester and 181 mg of glycine. The mixture was heated to 150° C. for 10minutes using a CEM Discover microwave reactor (City, State). Theresultant solution was cooled, and acidified with 1 N HCl solution toproduce a yellow precipitate. The precipitate was collected by filteringthe solution through a (medium) porous buchner filter funnel, andtriturated with methanol to give 68 mg of product; MS (−): m/z 353.02(M−1).

Example D-126[(4-Mercapto-7-trifluoromethyl-isoquinoline-3-carbonyl)-amino]-aceticacid a) 4-Hydroxy-7-trifluoromethyl-isoquinoline-3-carboxylic acid butylester

The title compound was prepared from was prepared from4-trifluoromethylphthalic acid under conditions analogous to ExampleD-7a-f; MS (+) m/z 314.1 (M+1)

b) [(4-Mercapto-7-trifluoromethyl-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was prepared from4-Hydroxy-7-trifluoromethyl-isoquinoline-3-carboxylic acid butyl esterunder conditions analogous to Example D-125; MS (−) m/z 328.33 (M−1)

Example D-127{[7-(4-Benzenesulfonylamino-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was synthesized from4-Hydroxy-7-(4-nitro-phenoxy)-isoquinoline-3-carboxylic acid butylester, Example D-124a, in analogy to examples D-123b-d substitutingbenzenesulfonyl chloride for p-toluenesulfonyl chloride in step c; MS(+): m/z 494.09 (M+1).

Example D-128{[4-Hydroxy-7-(4-methanesulfonylamino-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was synthesized from4-hydroxy-7-(4-nitro-phenoxy)-isoquinoline-3-carboxylic acid butylester, Example D-124a, in analogy to examples D-123b-d substitutingmethanesulfonyl chloride for p-toluenesulfonyl chloride in step c; MS(−): m/z 430.03 (M−1).

Example D-129{[7-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-(4-Chloro-phenoxy)-phthalonitrile

Prepared in analogy to Example D-88 a). ¹H NMR (200 MHz, DMSO) δ 8.09(d, J=9 Hz, 1H), 7.83 (d, J=2.6, 1H), 7.53 (d, J=8.6 Hz, 2H), 7.42 (dd,J=2.8, 8.6 Hz, 1H), 7.24 (d, J=8.6, 2H).

b) 4-(4-Chloro-phenoxy)-phthalic acid

Prepared in analogy to Example D-1 a). MS-(−)-ion: M−1=291.0.

c) [5-(4-Chloro-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid butyl ester

Prepared in analogy to Example D-100 c). ¹H NMR (200 MHz, DMSO) δ 7.48(d, J=8.6 Hz, 1H), 7.59 (d, J=9.0 Hz, 2H), 7.46 (m, 2H), 7.29 (d, J=9.0Hz, 2H), 4.46 (s, 2H), 4.16 (t, J=6.2 Hz, 2H), 1.61 (m, 2H), 1.38 (m,2H), 0.92 (t, J=7.0 Hz, 3H).

d) 6- and 7-(4-Chloro-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-1d). Mixture of two isomers.MS-(−)-ion: M−1=386.1.

e) 1-Chloro-6- and7-(4-chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester

Prepared in analogy to Example D-43 d). Mixture of two isomers.MS-(−)-ion: M−1=404.2.

f) 6- and 7-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example D-1 f). The two isomers were separated togive 7-(4-chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (Compound D-129A): MS-(−)-ion: M−1=370.3 and6-(4-chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (Compound D-129B): MS-(−)-ion: M−1=370.3.

g){[7-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from7-(4-chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (compound D-129A). MS-(−)-ion: M−1=371.0.

Example D-130{[6-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a){[6-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from6-(4-Chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (compound D-129B). MS-(−)-ion: M−1=371.1.

Example D-131{[6-(3-Fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-(3,4-Difluoro-phenoxy)-phthalonitrile

Prepared in analogy to Example D-88 a). ¹H NMR (200 MHz, DMSO) δ 8.14(d, J=9 Hz, 1H), 7.95 (d, J=2.6, 1H), 7.56 (dd, J=2.6, 8.6 Hz, 1H), 7.19(dt, J=2.4, 9.2 Hz, 1H), 7.04 (m, 2H).

b) 4-(3-Fluoro-5-methoxy-phenoxy)-phthalic acid

Prepared in analogy to Example D-1 a). One of the fluoro group issubstituted by a methoxy group during the hydrolysis. MS-(−)-ion:M−1=305.0.

c)[5-(3-Fluoro-5-methoxy-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid butyl ester

Prepared in analogy to Example D-100 c). ¹H NMR (200 MHz, DMSO) δ 7.93(d, J=8.6 Hz, 1H), 7.43 (m, 2H), 6.79-6.63 (m, 3H), 4.41 (s, 2H), 4.10(t, J=6.2, 2H), 1.54 (m, 2H), 1.30 (m, 2H), 0.86 (t, J=7.0, 3H).

d) 6- and7-(3-Fluoro-5-methoxy-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-1d). Mixture of two isomers.MS-(−)-ion: M−1=400.1.

e) 1-Chloro-6- and7-(3-fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example D-43 d). Mixture of two isomers.MS-(−)-ion: M−1=418.3.

f) 6- and7-(3-Fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

To a solution of 1-chloro-6- and7-(3-fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (176 mg) in ethyl acetate (3 ml) was added 10% Pd/C (50%wet, 70 mg) and then ammonium formate (264 mg). Resulting mixture washeated to reflux for 0.5 h. After cooling, the reaction mixture wasdiluted with ethyl acetate and filtered through a pad of Celite.Filtrate was concentrated and separated by chromatography to give 64 mg7-(3-fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (Compound D-131A) and 74 mg6-(3-fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (Compound D-131B): ¹H NMR (200 MHz, CD₃OD) δ 8.73 (s, 1H),8.15 (d, J=9.0 Hz, 1H), 7.71 (s, 1H), 7.59 (m, 1H), 6.65-6.47 (m, 3H),4.49 (t, J=6.6 Hz, 2H), 3.81 (s, 3H), 1.87 (m, 2H), 1.56 (m, 2H), 1.03(t, J=7.4. 3H).

g){[6-(3-Fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from6-(3-fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (compound D-131B). MS-(−)-ion: M−1=385.1.

Example D-132{[7-(3-Fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from7-(3-fluoro-5-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (compound D-131A). MS-(−)-ion: M−1=385.1.

Example D-133{[7-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a) 5-(3,4-Difluoro-phenoxy)-isoindole-1,3-dione

3,4-Difluorophenol (650 mg) was azeotroped with benzene and dissolved insodium methoxide solution in methanol (0.5 M, 10 ml). The methanol wasthen removed under reduced pressure under nitrogen. Then an anhydrousDMF (10 ml) solution of 4-nitrophthalimide (769 mg) was added to theprevious mixture. The resulting mixture was refluxed under nitrogen for23 h. The reaction was cooled down and added 80 ml water. The resultingprecipitate was filtered, washed with water (4×) and dried to give thetitle compound 685 mg. MS-(−)-ion: M−1=274.3.

b) [5-(3,4-Difluoro-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

To a pressure tube was added5-(3,4-difluoro-phenoxy)-isoindole-1,3-dione (680 mg), potassiumcarbonate (1 g), 3-pentanone (20 ml), and methyl bromoacetate (295 μL).The resulting mixture was heated to 105° C. for 17 h. The reaction wasdiluted with 20 ml water and extracted with ethyl acetate (2×). Theorganic layer was dried and concentrated. The mixture was purifiedthrough silica gel chromatography with 4:1 hexanes/ethyl acetate and 3:1hexanes/ethyl acetate to give 657 mg of the title compound.): ¹H NMR(200 MHz, DMSO) δ 7.95 (d, J=9.0 Hz, 1H), 7.64-7.41 (m, 4H), 7.15-7.08(m, 1H), 4.44 (s, 2H), 3.70 (s, 3H).

c) 6- and7-(3,4-Difluoro-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example D-1d). Mixture of two isomers.MS-(−)-ion: M−1=388.1.

d) 1-Chloro-6- and7-(3,4-difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester

Prepared in analogy to Example D-43 d). Mixture of two isomers wasdirectly carried on to next step.

e) 6- and 7-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-131 f). The two isomers were separatedto give 7-(3,4-difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound D-133A) and6-(3,4-difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (Compound 133B).

f){[7-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from7-(3,4-difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (compound D-133A). MS-(−)-ion: M−1=373.2.

Example D-134{[6-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a){[6-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from6-(3,4-difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (compound D-133B). MS-(−)-ion: M−1=373.2.

Example D-135{[4-Hydroxy-7-(4-trifluoromethoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 5-(4-Trifluoromethoxy-phenoxy)-isoindole-1,3-dione

Prepared in analogy to Example D-133 a). MS-(−)-ion: M−1=322.3.

b)[1,3-Dioxo-5-(4-trifluoromethoxy-phenoxy)-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

Prepared in analogy to Example D-133 b) by refluxing overnight. ¹H NMR(200 MHz, CDCl₃) δ 7.83 (d, J=8.6, 1H), 7.34-7.24 (m, 4H), 7.09 (d,J=8.6, 2H), 4.42 (s, 2H), 3.76 (s, 3H).

c) 1,4-Dihydroxy-6- and7-(3-trifluoromethoxy-phenoxy)-isoquinoline-3-carboxylic acid butylester

Prepared in analogy to Example D-1d). Mixture of two isomers.MS-(−)-ion: M−1=436.2.

d) 1-Chloro-4-hydroxy-6- and7-(3-trifluoromethoxy-phenoxy)-isoquinoline-3-carboxylic acid butylester

Prepared in analogy to Example D-43 d) by using microwave reactor withtoluene as solvent and with 1.5 equivalent of POCl₃. Mixture of twoisomers was directly carried on to next step.

e) 4-Hydroxy-6- and7-(3-trifluoromethoxy-phenoxy)-isoquinoline-3-carboxylic acid butylester

Prepared in analogy to Example D-131 f). The two isomers were separatedto give4-hydroxy-7-(3-trifluoromethoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester (Compound D-135A): MS-(+)-ion: M+1=422.2 and4-hydroxy-6-(3-trifluoromethoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester (Compound D-135B): MS-(−)-ion: M−1=420.6.

f){[4-Hydroxy-7-(4-trifluoromethoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from4-hydroxy-7-(3-trifluoromethoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester (compound D-135A). MS-(−)-ion: M−1=421.2.

Example D-136{[4-Hydroxy-6-(4-trifluoromethoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from4-hydroxy-6-(3-trifluoromethoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester (Compound D-135B). MS-(−)-ion: M−1=421.1.

Example D-137{[7-(3,5-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a)[5-(3,5-Difluoro-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester

To an 80 mL microwave reaction vessel was added(5-nitro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid ethyl ester (2g), 3,5-difluorophenol (1.12 g), potassium carbonate (1.39 g), anddimethyl acetamide (27 mL). The resulting mixture was reacted in themicrowave at 100° C. for 10 min. Water (280 mL) was added and theresulting precipitate was filtered, washed with water and dried. Furtherpurification by silica gel chromatography generated 0.94 g of the titlecompound. ¹H NMR (200 MHz, CDCl₃) δ 7.86 (d, J=8.2 Hz, 1H), 7.41-7.31(m, 2H), 6.67-6.57 (m, 3H), 4.41 (s, 2H), 4.22 (q, J=7.1 Hz, 2H), 1.29(t, J=7.0 Hz, 3H).

b) 6- and7-(3,5-Difluoro-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester

The title product was prepared in analogy to Example D-1d). Mixture oftwo isomers resulted. MS-(+)-ion: M+1=390.1.

c) 1-Chloro-6- and7-(3,5-difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester

Prepared in analogy to Example D-43 d) except that reaction was carriedout in microwave reactor at 135° C. for 10 min, using toluene as solventand 1.5 eq. POCl₃. Mixture of two isomers resulted. MS-(−)-ion:M−1=406.2.

d) 6- and 7-(3,5-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-131 f). The two isomers were separatedto give 7-(3,5-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound D-137A): MS-(−)-ion: M−1=372.2 and6-(3,5-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (Compound D-137B): MS-(+)-ion: M+1=374.1.

e){[7-(3,5-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from7-(3,5-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (compound D-137A). MS-(−)-ion: M−1=373.1.

Example D-138{[6-(3,5-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a){[6-(3,5-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from6-(3,5-difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (compound D-137B). MS-(−)-ion: M−1=373.1.

Example D-139({7-[4-(4-Fluoro-phenoxy)-phenoxy]-4-hydroxy-isoquinoline-3-carbonyl}-amino)-aceticacid a){5-[4-(4-Fluoro-phenoxy)-phenoxy]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-aceticacid ethyl ester

Prepared in analogy to Example D-137 a) by reacting(5-nitro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid ethyl esterwith 4-(4-fluoro-phenoxy)-phenol. ¹H NMR (200 MHz, CDCl₃) δ 7.80 (d,J=8.0 Hz, 1H), 7.31 (m, 2H), 7.06-7.01 (m, 8H), 4.39 (s, 2H), 4.21 (q,J=7.2, 2H), 1.30 (t, J=7.3, 3H).

b) 6- and7-[4-(4-Fluoro-phenoxy)-phenoxy]-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-1d). Mixture of two isomers resulted.MS-(−)-ion: M−1=462.1.

c) 1-Chloro-6- and7-[4-(4-fluoro-phenoxy)-phenoxy]-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-137 c). Mixture of two isomersresulted. MS-(+)-ion: M+1=482.1.

d) 6- and7-[4-(4-Fluoro-phenoxy)-phenoxy]-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-131 f). The two isomers were separatedto give7-[4-(4-Fluoro-phenoxy)-phenoxy]-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound D-139A): MS-(+)-ion: M+1=448.1 and6-[4-(4-fluoro-phenoxy)-phenoxy]-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound D-139B): MS-(+)-ion: M+1=448.2.

e)({7-[4-(4-Fluoro-phenoxy)-phenoxy]-4-hydroxy-isoquinoline-3-carbonyl}-amino)-aceticacid

The title product was prepared in analogy to Example D-37 e) startingfrom7-[4-(4-fluoro-phenoxy)-phenoxy]-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (compound D-139A). MS-(−)-ion: M−1=447.1.

Example D-140({6-[4-(4-Fluoro-phenoxy)-phenoxy]-4-hydroxy-isoquinoline-3-carbonyl}-amino)-aceticacid a)({7-[4-(4-Fluoro-phenoxy)-phenoxy]-4-hydroxy-isoquinoline-3-carbonyl}-amino)-aceticacid

The title product was prepared in analogy to Example D-37 e) startingfrom and6-[4-(4-fluoro-phenoxy)-phenoxy]-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound D-139B): MS-(−)-ion: M−1=447.1.

Example D-141{[7-(3-Chloro-4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a) 5-(3-Chloro-4-fluoro-phenoxy)-isoindole-1,3-dione

The title product was prepared in analogy to Example D-133 a).MS-(−)-ion: M−1=290.5.

b)[5-(3-Chloro-4-fluoro-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

The title product was prepared in analogy to Example D-133 b). ¹H NMR(200 MHz, CDCl₃) δ 7.83 (d, J=8.2 Hz, 1H), 7.32-7.14 (m, 4H), 6.99 (m,1H), 4.42 (s, 2H), 3.77 (s, 3H).

c) 6- and7-(3-Chloro-4-fluoro-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-1d). Mixture of two isomers.MS-(−)-ion: M−1=404.1.

d)1-Chloro-7-(3-chloro-4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-137 c). Mixture of two isomers.MS-(−)-ion: M−1=422.2.

e) 6- and7-(3-Chloro-4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example D-1 f). The two isomers were separated togive 7-(3-chloro-4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound D-141A): ¹H NMR (200 MHz, CDCl₃) δ 11.91 (s,1H), 8.64 (s, 1H), 8.38 (d, J=9.0 Hz, 1H), 7.46 (d, J=9.4, 1H),7.24-7.16 (m, 3H), 7.04-6.98 (m, 1H), 4.50 (t, J=6.8, 2H), 1.88 (q,J=7.2, 2H), 1.58-1.40 (m, 2H), 0.99 (t, J=7.2, 3H); and6-(3-Chloro-4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (Compound D-141B). MS-(+)-ion: M+1=390.1.

f){[7-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from7-(3-chloro-4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (Compound D-141A). MS-(−)-ion: M−1=389.0.

Example D-142{[6-(3-Chloro-4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a){[6-(3,4-Difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from6-(3-chloro-4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (Compound D-141B). MS-(−)-ion: M−1=389.0.

Example D-143{[7-(4-Chloro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-aceticacid a) 6- and7-(4-Chloro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example D-87 b) starting from a mixture of1-chloro-6- and 7-(4-chloro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (prepared as in example D-129 e). However, the pHadjustment was omitted. The two isomers were separated to give7-(4-chloro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylic acidbutyl ester (compound of example D-143 a) MS-(+)-ion M−1=386.1 and6-(4-chloro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylic acidbutyl ester (compound of example D-143 b) MS-(+)-ion M−1=386.1.

b){[7-(4-Chloro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from7-(4-chloro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylic acidbutyl ester (compound of example D-143 a) and reacting in a pressuretube overnight at 90 degree. MS-(−)-ion M−1=385.0.

Example D-144{[6-(4-Chloro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-aceticacid a){[6-(4-Chloro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from6-(4-chloro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylic acidbutyl ester (compound of example D-143 b) and reacting in a pressuretube overnight at 90 degree. MS-(−)-ion M−1=385.0.

Example D-145{[7-(3,5-Difluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-aceticacid a) 6- and7-(3,5-Difluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-87 b) starting from a mixture of1-chloro-6- and7-(3,5-difluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (prepared as in example D-137 c). The work-up procedure wasslightly different in omitting the pH adjustment. The two isomers wereseparated to give7-(3,5-difluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylicacid butyl ester (compound D-145 a1) MS-(−)-ion M−1=386.3 and6-(3,5-difluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylicacid butyl ester (compound D-145 a2) MS-(−)-ion M−1=386.3.

b){[7-(3,5-Difluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-37 e) starting from7-(3,5-difluoro-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylicacid butyl ester (compound D-145 a1) and reacting in a pressure tubeovernight at 90 degree. MS-(−)-ion M−1=387.1.

Example D-146{[4-Hydroxy-7-(4-methoxy-phenoxy)-1-methyl-isoquinoline-3-carbonyl]-amino}-aceticacid a. 6- and7-(4-Methoxy-phenoxy)-1-bromo-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

A mixture of 6- and7-(4-methoxy-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acid butylester (Compound D-88 d) (3.0 g) and phosphorus oxybromide (3.4 g) inanhydrous toluene (40 ml) was heated in a microwave reactor (sealedtube) for 15 min at 130° C. After cooling, reaction mixture wasconcentrated and saturated sodium bicarbonate aqueous solution (100 ml)was added. Stirred for 20 min and then extracted with ethyl acetate(2×100 ml). Combined organic layers were washed with water, brine, driedover magnesium sulfate, filtered and concentrated to give the titlecompound (3.1 g). MS-(+)-ion M+1=446.05, 448.05.

b. 6- and7-(4-Methoxy-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylic acidbutyl ester

A mixture of 6- and7-(4-methoxy-phenoxy)-1-bromo-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (232 mg), Pd(PPh₃)₄ (60 mg), trimethylboroxine (65 mg) andpotassium carbonate (216 mg) in dioxane (4 ml) was heated in a microwavereactor (sealed tube) for 10 min at 120° C. After cooling, the reactionmixture was diluted with water (15 ml). Acidified by 2 N HCl to pH=4.Extracted with ethyl acetate. Organic layer was washed with brine, driedover magnesium sulfate and filtered. Filtrated was concentrated andseparated by silica gel chromatography (eluting with 25% to 50% ethylacetate in hexanes) to give7-(4-methoxy-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylic acidbutyl ester (35 mg) (Compound D-146 b1) (MS-(+)-ion M+1=382.18) and6-(4-methoxy-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylic acidbutyl ester (61 mg) (Compound D-146 b2) (MS-(+)-ion M+1=382.16).

c){[4-Hydroxy-7-(4-methoxy-phenoxy)-1-methyl-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared from7-(4-methoxy-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylic acidbutyl ester (Compound D-146 b1) in analogy to Example D-107 b)(microwave reaction temperature 120° C., reaction time 10 min).MS-(−)-ion M−1=381.09.

Example D-147{[4-Hydroxy-6-(4-methoxy-phenoxy)-1-methyl-isoquinoline-3-carbonyl]amino}-aceticacid

Prepared from6-(4-methoxy-phenoxy)-4-hydroxy-1-methyl-isoquinoline-3-carboxylic acidbutyl ester (Compound D-146 b2) in analogy to Example D-146 c).MS-(−)-ion M−1=381.10.

Example D-148[(6-Cyclohexyloxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acida. (5-Hydroxy-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid ethylester

Prepared in analogy to example D-100 c) from 4-hydroxy-phthalic acid andglycine ethyl ester HCl salt. ¹H NMR (200 MHz, DMSO-d₆) δ 11.0 (br s,1H), 7.74 (d, J=7.8 Hz, 1H), 7.17 (m, 2H), 4.35 (s, 2H), 4.13 (q, J=7.0Hz, 2H), 1.20 (t, J=7.0 Hz, 3H).

b. (5-Cyclohexyloxy-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acidethyl ester

To a mixture of (5-hydroxy-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-aceticacid ethyl ester (8.0 g) in anhydrous tetrahydrofuran (160 ml) was addedcyclohexanol (3.2 g), diethylazadicarboxylate (6.9 g) and then triphenylphosphine (12.6 g). Resulting mixture was stirred at room temperatureovernight and concentrated. Residue was partitioned between water andethyl acetate. Aqueous layer was extracted with ethyl acetate. Combinedorganic layers were washed with brine, dried over magnesium sulfate andfiltered. Filtrate was concentrated and purified by silica gelchromatography (eluting with 5% ethyl acetate in methylene chloride) togive the title compound (6.2 g). ¹H NMR (200 MHz, CDCl₃) δ 7.73 (dd,J=8.2, 0.8 Hz, 1H), 7.30 (br s, 1H), 7.12 (m, 1H), 4.38 (m, 3H), 4.21(q, J=7.1 Hz, 2H), 2.02 (m, 2H), 1.82-1.25 (m, 13H).

c. 6- and 7-Cyclohexyloxy-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example D-1 d) to give7-cyclohexyloxy-1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound D-148 c1) (MS-(+)-ion M+1=360.16) and6-cyclohexyloxy-1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound D-148 c2) (MS-(+)-ion M+1=360.18).

d. 1-Bromo-6-cyclohexyloxy-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example D-146 a) from6-cyclohexyloxy-1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound D-148 c2). MS-(+)-ion M+1=422.10, 424.10.

e. 6-Cyclohexyloxy-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

To a mixture of1-bromo-6-cyclohexyloxy-4-hydroxy-isoquinoline-3-carboxylic acid butylester (1.0 g) in ethyl acetate (20 ml) was added 10% Pd/C (50% wet) (460mg) and then ammonium formate (1.5 g). Resulting mixture was refluxedfor 4 h. After cooling, reaction mixture was filtered and concentrated.The residue was purified by silica gel chromatography (5%-10% ethylacetate in methylene chloride) to give the title compound (640 mg).MS-(+)-ion M+1=344.22.

f. [(6-Cyclohexyloxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared in analogy to Example D-146 c). MS-(−)-ion M−1=343.15.

Example D-149[(7-Cyclohexyloxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acida. 1-Bromo-7-cyclohexyloxy-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example D-146 a) from7-cyclohexyloxy-1,4-dihydroxy-isoquinoline-3-carboxylic acid butyl ester(Compound D-148 c1). MS-(+)-ion M+1=422.12, 424.12.

b. 7-Cyclohexyloxy-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

Prepared in analogy to Example D-148 e). MS-(+)-ion M+1=344.22.

c. [(7-Cyclohexyloxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared in analogy to Example D-146 c). MS-(−)-ion M−1=343.17.

Example D-150[(7-Cyclohexyloxy-4-hydroxy-1-methyl-isoquinoline-3-carbonyl)-amino]-aceticacid a. 7-Cyclohexyloxy-4-hydroxy-1-methyl-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-146 b). MS-(+)-ion M+1=358.21.

b.[(7-Cyclohexyloxy-4-hydroxy-1-methyl-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared in analogy to Example D-146 c). MS-(+)-ion M+1=359.15.

Example D-151[(7-Cyclohexylsulfanyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a.(5-Cyclohexylsulfanyl-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acidethyl ester

A mixture of 5-nitro-isoindole-1,3-dione (10.0 g), cyclohexanethiol (9.1g) and potassium carbonate (18.7 g) in acetone (260 ml) was heated toreflux overnight. After cooling, the mixture was diluted with water (250ml) and then acidified by 6 N HCl to pH=4. Precipitate was collected anddried in vacuo to give the intermediate5-cyclohexylsulfanyl-isoindole-1,3-dione (15.6 g). This intermediate wasdissolved in acetone (170 ml) and to the mixture was added bromoethylacetate (10.6 g) and potassium carbonate (23.8 g). The mixture wasrefluxed overnight. After cooling, reaction mixture was filtered andrinsed with ethyl acetate. Filtrate was concentrated and purified bysilica gel chromatography (10%-50% ethyl acetate in methylene chloride)to give the title compound (13.1 g). ¹H NMR (200 MHz, CDCl₃) δ 7.73 (m,2H), 7.56 (dd, J=7.8, 1.6 Hz, 1H), 4.40 (s, 2H), 4.21 (q, J=7.0 Hz, 2H),3.37 (m, 1H), 2.07-1.28 (m, 13H).

b. 6- and 7-Cyclohexylsulfanyl-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-21 b). MS-(+)-ion M+1=376.20.

c. 6- and7-Cyclohexylsulfanyl-1-chloro-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

A mixture of 6- and7-cyclohexylsulfanyl-1,4-dihydroxy-isoquinoline-3-carboxylic acid butylester (1.0 g) and phosphorus oxychloride (491 mg) in anhydrous toluene(14 ml) was heated in a microwave reactor (sealed tube) (180° C., 30min). After cooling, reaction mixture was quenched with saturated sodiumbicarbonate. Stirred at room temperature for 20 min. and extracted withethyl acetate twice. Combined organic layers were washed with water,brine, dried over magnesium sulfate, filtered, and concentrated to givethe title compound (0.5 g). MS-(+)-ion M+1=394.12.

d. 6- and 7-Cyclohexylsulfanyl-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared in analogy to Example D-1 f) to give7-cyclohexylsulfanyl-4-hydroxy-isoquinoline-3-carboxylic acid butylester (128 mg) (Compound D-151 d1) (MS-(+)-ion M+1=360.15) and6-cyclohexylsulfanyl-4-hydroxy-isoquinoline-3-carboxylic acid butylester (130 mg) (Compound D-151 d2) (MS-(+)-ion M+1=360.17).

e)[(7-Cyclohexylsulfanyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared in analogy to Example D-1 g) from7-cyclohexylsulfanyl-4-hydroxy-isoquinoline-3-carboxylic acid butylester (Compound D-151 d1). MS-(−)-ion M−1=359.11.

Example D-152[(7-Cyclohexanesulfonyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 7-Cyclohexanesulfonyl-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

A mixture of 7-cyclohexylsulfanyl-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (Compound D-151 d1) (64 mg) and m-chloroperoxybenzoicacid (111 mg) in methylene chloride (2 ml) was stirred at roomtemperature overnight. It was diluted with methylene chloride (50 ml)and washed successively with saturated sodium bicarbonate aqueoussolution (2×50 ml), water, and brine. The organic layer was dried overmagnesium sulfate and filtered. Filtrate was concentrated and purifiedby silica gel chromatography (eluting with 3%-15% ethyl acetate inmethylene chloride) to give the title compound (70 mg). MS-(+)-ionM+1=392.20.

b.[(7-Cyclohexanesulfonyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared in analogy to Example D-146 c). MS-(−)-ion M−1=391.05.

Example D-153[(4-Hydroxy-1-isobutyl-isoquinoline-3-carbonyl)-amino]-acetic acid a.4-Benzyloxy-1-isobutyl-isoquinoline-3-carboxylic acid butyl ester

A mixture of 4-Benzyloxy-1-bromo-isoquinoline-3-carboxylic acid butylester (207 mg, 0.5 mmol, see example D-86a), Pd(PPh₃)₄ (58 mg, 0.05mmol), 2-methylpropylboronic acid (78 mg, 0.75 mmol), K₂CO₃ (207 mg, 1.5mmol), and 1,4-dioxane (4 ml) was refluxed with stirring for 48 h.Subsequently, the mixture was concentrated in vacuo. To the residue wasadded water (5 ml) and the mixture was extracted with EtOAc (2×20 ml).The organic phase was dried over MgSO₄ and evaporated in vacuo.Purification of the residue by flash column chromatography on silica gelusing hexanes EtOAc=88 12 as the eluent gave the title compound as ayellowish oil (136 mg); MS-(+)-ion M+1=392.3.

b) 4-Hydroxy-1-isobutyl-isoquinoline-3-carboxylic acid butyl ester

A mixture of 4-Benzyloxy-1-isobutyl-isoquinoline-3-carboxylic acid butylester (125 mg, 0.32 mmol), Pd/C (50 mg, Aldrich, 10 wt % Pd) and EtOAc(15 ml) were stirred at ambient pressure and temperature under an H₂atmosphere for 24 h. The mixture was then filtered through a pad ofcelite. Concentration of the filtrate in vacuo yielded the titlecompound as a yellowish oil (87 mg); MS-(+)-ion M+1=302.2.

c) [(4-Hydroxy-1-isobutyl-isoquinoline-3-carbonyl)-amino]-acetic acid

Synthesized from 4-Hydroxy-1-isobutyl-isoquinoline-3-carboxylic acidbutyl ester in analogy to example D-1g); MS-(+)-ion M+1=303.2.

Example D-154[(4-Hydroxy-1-pyridin-2-yl-isoquinoline-3-carbonyl)-amino]-acetic acida. 4-Benzyloxy-1-pyridin-2-yl-isoquinoline-3-carboxylic acid butyl ester

To a solution of Pyridin-2-ylboronic acid (323 mg, 2.5 mmol) in EtOH(2.5 ml) was added subsequently toluene (15 ml),4-Benzyloxy-1-bromo-isoquinoline-3-carboxylic acid butyl ester (1.035mg, 2.5 mmol, see example D-86a), Pd(PPh₃)₄ (292 mg, 0.25 mmol), and aq.2 M Na₂CO₃ solution (2.5 ml, 5 mmol). The mixture was then refluxed withstirring under N₂ protection for 24 h. Subsequently, the mixture wasconcentrated in vacuo. To the residue was added water (15 ml) and themixture was extracted with EtOAc (30 ml). The organic phase was driedover MgSO₄ and evaporated in vacuo. Purification of the residue by flashcolumn chromatography on silica gel using CH₂Cl₂ MeOH=98 2 as the eluentgave a dark oil that was further purified by flash column chromatographyon silica gel using CH₂Cl₂ MeOH=99 1 as the eluent and subsequently bypreparative TLC using CH₂Cl₂ MeOH=98 2 as the eluent (had to be repeatedseveral times) to give the title compound as a yellow oil (19 mg);MS-(+)-ion M+1=413.2.

b) 4-Hydroxy-1-pyridin-2-yl-isoquinoline-3-carboxylic acid butyl ester

Synthesized from 4-Benzyloxy-1-pyridin-2-yl-isoquinoline-3-carboxylicacid butyl ester in analogy to example D-153b); MS-(−)-ion M−1=321.4.

c) [(4-Hydroxy-1-pyridin-2-yl-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from 4-Hydroxy-1-pyridin-2-yl-isoquinoline-3-carboxylic acidbutyl ester in analogy to example D-1g); MS-(+)-ion M+1=324.1.

Example D-155[(1-Ethyl-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a. 4-Hydroxy-7-phenoxy-1-vinyl-isoquinoline-3-carboxylic acid butylester

A mixture of 1-Bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester (416 mg, 1 mmol, see example D-28a), Pd(PPh₃)₄ (118 mg, 0.1mmol), 2,4,6-Trivinylcyclotriboroxane-pyridine complex (241 mg, 1 mmol),K₂CO₃ (414 mg, 3 mmol), and 1,4-dioxane (8 ml) was refluxed withstirring under N₂ protection for 3 h. Subsequently, the mixture wasconcentrated in vacuo. To the residue was added water (5 ml) and themixture was extracted with EtOAc (20 ml). The organic phase was driedover MgSO₄ and evaporated in vacuo. Purification of the residue by flashcolumn chromatography on silica gel using hexanes EtOAc=98 2 as theeluent gave the title compound as a yellowish solid (65 mg); MS-(+)-ionM+1=364.1.

b) 1-Ethyl-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butylester

Synthesized from 4-Hydroxy-7-phenoxy-1-vinyl-isoquinoline-3-carboxylicacid butyl ester in analogy to example D-153b); MS-(+)-ion M+1=366.1.

c) [(1-Ethyl-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from 1-Ethyl-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylicacid butyl ester in analogy to example D-1g); MS-(+)-ion M+1=367.1.

Example D-156[(1-Dimethylaminomethyl-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid a.1-Dimethylaminomethyl-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester

A mixture of 4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acidbutyl ester (177 mg, 0.5 mmol; see example D-1f),N,N-dimethylmethyleneammonium iodide (94 mg, 0.5 mmol), K₂CO₃ (104 mg,0.75 mmol), and anhydrous CH₂Cl₂ (3 ml) was stirred at ambienttemperature for 2.5 d before the mixture was concentrated in vacuo. Tothe residue was added water (15 ml), the mixture was acidified byaddition of 6 N HCl and then washed with Et₂O (3×30 ml). Subsequently,the mixture was neutralized by the addition of concentrated aqueousNaHCO₃ and extracted with EtOAc (20 ml). The organic phase was driedover MgSO₄ and concentrated in vacuo to give the title compound as adark oil (34 mg); MS-(+)-ion M+1=411.1.

b)[(1-Dimethylaminomethyl-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid

Synthesized from1-Dimethylaminomethyl-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester in analogy to example D-1g); MS-(+)-ion M+1=412.0.

Example D-157[(4-Hydroxy-1-methyl-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid a) 1-Bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylicacid

1,4-Dihydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butylester (Example D-1 d) Compound A) (29.0 g) and phosphorous oxybromide(67.5 g) in 600 ml anhydrous acetonitrile was stirred at reflux for 4hours. After cooling the reaction mixture was concentrated and saturatedsodium bicarbonate solution and ethyl acetate were added to the residueand stirred overnight. Precipitate that formed between layers wascollected and washed with water to give the title compound (10.2 g).MS-(+)-ion M+1=376.0, 378.1.

b) 1-Bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acidmethyl ester

1-Bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid (10.0g), potassium carbonate (3.7 g) and methyl sulfate (3.4 g) weresuspended in 500 ml acetone and stirred at reflux overnight. Reactionmixture was concentrated and residue partitioned between 1 Nhydrochloric acid and ethyl acetate. Organic layer was dried overmagnesium sulfate and filtered. Filtrate concentrated to give titlecompound (9.6 g). MS-(+)-ion M+1=389.9, 391.9.

c) 4-Hydroxy-1-methyl-7-phenylsulfanyl-isoquinoline-3-carboxylic acidmethyl ester

1-Bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid methylester (0.2 g), tetrakis(triphenylphosphine)palladium (60 mg), trimethylboroxine (65 mg), and potassium carbonate in 1,4-dioxane (4 ml) wereheated in a microwave reactor (sealed tube) for 10 min at 140° C. Aftercooling reaction mixture was concentrated and partitioned between 1 Nhydrochloric acid and ethyl acetate. Organic layer dried over magnesiumsulfate and filtered. Filtrate concentrated and separated by silica gelchromatography (eluting with 2% ethyl acetate in methylene chloride) togive the title compound (47 mg). MS-(+)-ion M+1=326.1.

d)[(4-Hydroxy-1-methyl-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared in analogy to Example D-146 c). MS-(+)-ion M+1=369.1.

Example D-158{[4-Hydroxy-1-methyl-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a.4-Hydroxy-1-methyl-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to Example D-157 d) from4-hydroxy-1-chloro-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carboxylicacid butyl ester (of Example D-92 f). MS-(+)-ion M+1=420.2.

b){[4-Hydroxy-1-methyl-7-(4-trifluoromethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to Example D-146 c). MS-(+)-ion M+1=421.2.

1. A compound represented by formula:

wherein: R¹ is selected from the group consisting of hydrogen, halo,alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, substitutedamino, aminoacyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, and —XR⁶ where X isoxygen, —S(O)_(n)— or —NR⁷— where n is zero, one or two, R⁶ is selectedfrom the group consisting of alkyl, substituted alkyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic, and R⁷ is hydrogen, alkyl or aryl or, when X is —NR⁷—,then R⁷ and R⁶, together with the nitrogen atom to which they are bound,can be joined to form a heterocyclic or substituted heterocyclic group;and R² and R³ are independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, halo, hydroxy, cyano, —S(O)_(n)—N(R⁶)—R⁶ where nis 0, 1, or 2, —NR⁶C(O)NR⁶R⁶, and —XR⁶ where X is oxygen, —S(O)_(n)— or—NR⁷— where n is zero, one or two, each R⁶ is independently selectedfrom the group consisting of hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclicprovided that when X is —SO— or —SO₂—, then R₆ is not hydrogen, and R⁷is selected from the group consisting of hydrogen, alkyl, and aryl, orR², R³ together with the carbon atom pendent thereto, form an arylsubstituted aryl, heteroaryl, or substituted heteroaryl; or apharmaceutically acceptable salt, ester, or amide thereof; with theproviso that at least one of the following occurs: 1) R¹ is fluoro,bromo, iodo, alkyl, substituted alkyl, alkoxy, aminoacyl, substitutedalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic, or —XR⁶ where X is oxygen,—S(O)_(n)— or —NR⁷— where n is zero, one or two, R⁶ is selected from thegroup consisting of alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic, and R⁷ is hydrogen, alkyl or aryl; or 2) R² is substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,fluoro, iodo, cyano, or —XR⁶ where X is oxygen, —S(O)_(n)— or —NR⁷—where n is zero, one or two, R⁶ is selected from the group consisting ofalkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic, andR⁷ is hydrogen, alkyl or aryl provided that: a) when R² is substitutedalkyl such a substituent does not include trifluoromethyl; b) —XR⁶ isnot alkoxy; and c) when —XR⁶ is substituted alkoxy such a substituentdoes not include benzyl or benzyl substituted by a substituent selectedfrom the group consisting of (C₁-C₅) alkyl and (C₁-C₅) alkoxy or doesnot include a fluoroalkoxy substituent of the formula:—O—[CH₂]_(x)—C_(f)H_((2f+1−g))F_(g) where x is zero or one; f is aninteger of from 1 to 5; and g is an integer of from 1 to (2f+1); or 3)R³ is substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, bromo, iodo, or —XR⁶ where X is oxygen, —S(O)_(n)— or —NR⁷—where n is zero, one or two, R⁶ is selected from the group consisting ofalkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic, andR⁷ is hydrogen, alkyl or aryl provided that: a) when R³ is substitutedalkyl such a substituent does not include trifluoromethyl; b) —XR⁶ isnot alkoxy; and c) when —XR⁶ is substituted alkoxy such a substituentdoes not include benzyl or benzyl substituted by a substituent selectedfrom the group consisting of (C₁-C₅) alkyl and (C₁-C₅) alkoxy or doesnot include a fluoroalkoxy substituent of the formula:—O—[CH₂]_(x)—C_(f)H_((2f+1−g))F_(g) where x is zero or one; f is aninteger of from 1 to 5; and g is an integer of from 1 to (2f+1).
 2. Thecompound of claim 1, wherein: R² and R³ are independently selected fromthe group consisting of hydrogen and —XR⁶ where X is oxygen or sulfur,and each R⁶ is independently selected from the group consisting of aryland substituted aryl; with the proviso that at least one of thefollowing occurs: 1) R¹ is fluoro, bromo, iodo, alkyl, substitutedalkyl, alkoxy, aminoacyl, substituted alkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, or —XR⁶ where X is oxygen, —S(O)_(n)— or —NR⁷— where n iszero, one or two, R⁶ is selected from the group consisting of alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic, and R⁷ ishydrogen, alkyl or aryl; 2) R² is —XR⁶ where X is oxygen or sulfur, andR⁶ is selected from the group consisting of aryl and substituted aryl;or 3) R³ is —XR⁶ where X is oxygen or sulfur, and R⁶ is selected fromthe group consisting of aryl and substituted aryl.
 3. The compound ofclaim 1, wherein: R² and R³ are independently selected from the groupconsisting of hydrogen and —XR⁶ where X is oxygen or sulfur, each R⁶ isindependently selected from the group consisting of aryl, andsubstituted aryl; with the proviso that at least one of the followingoccurs: 1) R² is —XR⁶ where X is oxygen or sulfur and R⁶ is selectedfrom the group consisting of aryl and substituted aryl; or 2) R³ is —XR⁶where X is oxygen or sulfur and R⁶ is selected from the group consistingof aryl and substituted aryl.
 4. The compound of claim 1, wherein: R² is—XR⁶ where X is oxygen or sulfur, and R⁶ is aryl or aryl substitutedwith one or two substituents independently selected from the groupconsisting of halo, alkyl, alkyl substituted with up to three halosubstituents, alkoxy, alkoxy substituted with up to three halosubstituents, nitro, alkylsulfonamido, and arylsulfonamido; and R³ ishydrogen.
 5. The compound of claim 4, wherein R¹ is selected from thegroup consisting of hydrogen, halo and alkyl.
 6. A pharmaceuticalcomposition comprising one or more compounds of claim 1 or apharmaceutically acceptable salt, ester, or amide thereof and apharmaceutically acceptable carrier or excipient.
 7. The pharmaceuticalcomposition of claim 6, wherein the compound is of the formula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 8. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 9. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 10. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 11. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 12. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 13. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 14. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 15. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 16. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 17. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 18. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 19. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 20. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 21. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 22. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 23. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.
 24. Thepharmaceutical composition of claim 6, wherein the compound is of theformula:

or a pharmaceutically acceptable salt, ester, or amide thereof.